[go: up one dir, main page]

WO2016024205A1 - Oligomères ciblant l'expansion d'une répétition hexanucléotidique dans le gène c9orf72 humain - Google Patents

Oligomères ciblant l'expansion d'une répétition hexanucléotidique dans le gène c9orf72 humain Download PDF

Info

Publication number
WO2016024205A1
WO2016024205A1 PCT/IB2015/056080 IB2015056080W WO2016024205A1 WO 2016024205 A1 WO2016024205 A1 WO 2016024205A1 IB 2015056080 W IB2015056080 W IB 2015056080W WO 2016024205 A1 WO2016024205 A1 WO 2016024205A1
Authority
WO
WIPO (PCT)
Prior art keywords
gapmer
c90rf72
region
oligomer
rna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2015/056080
Other languages
English (en)
Other versions
WO2016024205A8 (fr
Inventor
Mads Aaboe JENSEN
Morten Lindow
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pfizer Corp Belgium
Pfizer Corp SRL
Original Assignee
Pfizer Corp Belgium
Pfizer Corp SRL
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pfizer Corp Belgium, Pfizer Corp SRL filed Critical Pfizer Corp Belgium
Priority to US14/826,829 priority Critical patent/US20160108396A1/en
Publication of WO2016024205A1 publication Critical patent/WO2016024205A1/fr
Publication of WO2016024205A8 publication Critical patent/WO2016024205A8/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/34—Allele or polymorphism specific uses

Definitions

  • Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are severe neurological diseases with no effective treatment.
  • FTD is a common cause of early-onset dementia and includes a group of disorders characterized clinically by abnormalities in behavior, language, and personality.
  • ALS is typified by degeneration of motor neurons leading to muscle atrophy and paralysis. Due to their substantial clinical and pathological overlap, FTD and ALS have been proposed to constitute a disease spectrum. For example, frontal lobe impairment is seen in ALS and some FTD patients develop features of motor neuron disease.
  • GGGGCC hexanucleotide repeat present in a non-coding region of the Homo sapiens chromosome 9 open reading frame 72 (C90RF72) gene (termed “hexanucleotide repeat expansion” or simply “repeat expansion,” as used interchangeably herein) as the most common genetic cause of ALS and FTD.
  • the C90RF72 gene encodes long and short protein isoforms of a protein of uncertain function (Ling et al., Neuron 79: 416-438 (2011)). Although the mechanism by which C90RF72 hexanucleotide repeat expansion causes the familial versions FTD and ALS remains to be elucidated, preliminary analysis suggests a variety of mechanisms may be at work. More recent studies found intracellular inclusions of aggregated di-peptide repeat proteins in the brains of patients with familial FTD or ALS, presumably generated by non-standard translation of the expanded GGGCC repeat. This observation suggests the hypothesis that the protein aggregates are neurotoxic and may in part explain the etiology of the diseases.
  • oligomers that are complementary to the C90RF72 gene.
  • the present disclosure provides gapmer compositions for targeting RNA containing a pathogenic number (e.g., at least 30 repeats) of hexanucleotide repeats transcribed from the C90RF72 gene and methods of treating subjects with disorders associated with such hexanucleotide repeat expansion.
  • a pathogenic number e.g., at least 30 repeats
  • a gapmer comprising between 12 to 30 nucleosides, complementary to at least a 12 contiguous nucleobase portion of SEQ ID NO: 187, wherein the gapmer further comprises in 5' to 3' order: (a) a 5' flanking region consisting of 1 to 5 contiguously linked nucleosides, at least one of which is an LNA monomer,
  • E2 The gapmer of E1 , wherein the expanded hexanucleotide region contains at least 20 hexanucleotide repeats.
  • E3. The gapmer of E1 , wherein the expanded hexanucleotide region contains at least 30 hexanucleotide repeats.
  • E4 The gapmer of E1 , wherein the expanded hexanucleotide region contains at least 50 hexanucleotide repeats.
  • E5 The gapmer of E1 , wherein the expanded hexanucleotide region contains at least 75 hexanucleotide repeats.
  • E6 The gapmer of E1 , wherein the expanded hexanucleotide region contains at least 100 hexanucleotide repeats.
  • E7 The gapmer of E1 or E2, wherein the IC50 for inhibition of expression of the C90RF72 sense transcript containing an expanded hexanucleotide repeat region is 50% or less of the IC50 for a wild-type C90RF72.
  • E8 The gapmer of E1 or E2, wherein the IC50 for inhibition of expression of the C90RF72 sense transcript containing an expanded hexanucleotide repeat region is 40% or less of the IC50 for a wild-type C90RF72.
  • E9 The gapmer of E1 or E2, wherein the IC50 for inhibition of expression of the C90RF72 sense transcript containing an expanded hexanucleotide repeat region is 30% or less of the IC50 for a wild-type C90RF72.
  • E10 The gapmer of E1 or E2, wherein the IC50 for inhibition of expression of the C90RF72 sense transcript containing an expanded hexanucleotide repeat region is 20% or less of the IC50 for a wild-type C90RF72.
  • E11 The gapmer of E1 or E2, wherein the IC50 for inhibition of expression of the C90RF72 sense transcript containing an expanded hexanucleotide repeat region is 10% or less of the IC50 for a wild-type C90RF72.
  • E12. The gapmer of E1 or E2, wherein the IC50 for inhibition of expression of the C90RF72 sense transcript containing an expanded hexanucleotide repeat region is 5% or less of the IC50 for a wild-type C90RF72.
  • E13 The gapmer of any of E1 to E12, which is capable of reducing the expression of the C90RF72 sense transcript containing an expanded hexanucleotide repeat region in a cell with an IC50 of less than 10 micromolar without electroporation.
  • E14 The gapmer of any of any of E1 to E13, having a sequence that is identical to at least a 12 nucleobase portion of a nucleobase sequence selected from the group consisting of SEQ I D NO: 1 to SEQ I D NO: 175. E15.
  • the gapmer of E6, having a sequence that is identical to at least a 12 contiguous nucleobase portion of a nucleobase sequence selected from the group consisting of SEQ ID NOs: 9, 10, 11 , 12, 13, 14, 16, 17, 19, 20, 21 , 22, 23, 24, 26, 95, 98, 103, 106, 109, 119, 120, 123, 125, 126, 127, 134, 135, 142, 144, 145, 146, 147, 149, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 166, 167, 169, 170, 172, 173, 174, and 175.
  • E16 The gapmer of E6, having a sequence that is identical to at least a 12 contiguous nucleobase portion of a nucleobase sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 20, 103, 106, 109, 149, 155, 158, 160 and 161.
  • E17 The gapmer of any of E1-E16, wherein the IC50 for the C90RF72 sense transcript and IC50 for the C90RF72 antisense transcript are less than 5 ⁇ .
  • E18 The gapmer of E17, having a sequence selected from the group consisting of SEQ ID NOs: 9, 12, 13, 14, 16, 17, 19, 20, 21 , 26, 103, 106, 109, 119, 126, 127, 135, 142, 144, 146, 147, 149, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 166, and 170.
  • E19 The gapmer of E17, having a sequence selected from the group consisting of SEQ ID NOs: 9, 13, 16, 17, 19, 106, 135, 142, 147, 154, 156, 157, 158, 159 and 162.
  • E20 The gapmer of any of the E1 to E19, wherein the gapmer sequence does not consist of any sequence selected from the group consisting of SEQ ID Nos:189 - 234.
  • E21 The gapmer of any of E1 - E7, which is complementary to an at least 12 contiguous nucleobase portion between nucleobase 5159 and 1 1702 of SEQ I D NO: 187, for example, the gapmer of E1 - E7, which is complementary to an at least 12 contiguous nucleobase portion between nucleobases 5311 - 5359 of SEQ ID NO: 187.
  • E22 The gapmer of any of the E1 to E21 which is not complementary to an at least 5 contiguous nucleobase portion of SEQ ID NO: 188.
  • the gapmer of E10, wherein the modified base is 5-methylcytosine.
  • the gap region of the gapmer of any of E1 - E8 comprises at least 5-methylcytosine.
  • E25 The gapmer of any of E1 - E1 1 , wherein all internucleoside linkages are phosphorothioate.
  • E26 The gapmer of any of E1 - E12, wherein all of the nucleosides in the 5' flanking region and 3' flanking region are modified.
  • E27 The gapmer of any of E1 - E13, wherein all modified nucleosides are LNA monomers.
  • E28 The gapmer of any of E1 - E14, wherein all LNA monomers are beta-D-oxy-LNA monomers.
  • E29 The gapmer of any of E1 - E15, wherein the nucleobase sequence of said gapmer is CCc egg ccc egg CC (SEQ ID NO: 12), wherein the 5' flanking region and 3' flanking region are each respectively indicated by capital letters; and wherein the gap region is indicated by lower case letters.
  • E30 The gapmer of E16, wherein at least one cytosine is 5-methylcytosine.
  • E31 The gapmer of E16 or E17, wherein cytosines at positions 1 , 2, 4, 10, 13, and 14 are 5-methylcytosine.
  • E32 The gapmer of any of E1 to E15, wherein the nucleobase sequence of said gapmer is GCc ccg gcc ccg gCC (SEQ ID NO: 13), wherein the 5' flanking region and 3' flanking region are each respectively indicated by capital letters, and wherein the gap region is indicated by lower case letters.
  • E33 The gapmer of E19, wherein at least one cytosine is 5-methylcytosine.
  • E34 The gapmer of E19 or E20, wherein cytosines at positions 2, 5, 1 1 , 14, and 15 are 5-methylcytosine.
  • E35 The gapmer of any of E1 to E15, wherein the nucleobase sequence of said gapmer is CCc egg ccc egg ccC C (SEQ ID NO: 14), wherein the 5' flanking region and 3' flanking region are each respectively indicated by capital letters, and wherein the gap region is indicated by lower case letters.
  • E36 The gapmer of E22, wherein at least one cytosine is 5-methylcytosine.
  • E37 The gapmer of E22 or E23, wherein cytosines at positions 1 , 2, 4, 10, 15, and 16 are 5-methylcytosine.
  • E38 The gapmer of any of E1 to E15, wherein the nucleobase sequence of said gapmer is Ggc ccc ggc ccC (SEQ ID NO:20), wherein the 5' flanking region and 3' flanking region are each respectively indicated by capital letters, and wherein the gap region is indicated by lower case letters.
  • E39 The gapmer of E25, wherein at least one cytosine is 5-methylcytosine.
  • E40 The gapmer of E25 or E26, wherein cytosines at positions 6 and 12 are 5- methylcytosine.
  • E41 The gapmer of any of E1 to E15, wherein the nucleobase sequence of said gapmer is CCg gcc ccg gcC CC (SEQ ID NO: 103), wherein the 5' flanking region and 3' flanking region are each respectively indicated by capital letters, and wherein the gap region is indicated by lower case letters.
  • E42 The gapmer of E28, wherein at least one cytosine is 5-methylcytosine.
  • E43 The gapmer of E28 or E29, wherein cytosines at positions 1 , 2, 8, 12, 13, and 14 are 5-methylcytosine.
  • E44 The gapmer of any of E1 to E43, wherein the nucleobase sequence of said gapmer is CCc ggc ccc ggC CC (SEQ ID NO: 106), wherein the 5' flanking region and 3' flanking region are each respectively indicated by capital letters; and wherein the gap region is indicated by lower case letters.
  • E45 The gapmer of E31 , wherein at least one cytosine is 5-methylcytosine.
  • E46 The gapmer of E31 or E32, wherein cytosines at positions 1 , 2, 3, 9, 12, 13, and 14 are 5-methylcytosine.
  • E47 The gapmer of any of E1 to E15, wherein the nucleobase sequence of said gapmer is GGc ccc ggc ccC GG (SEQ ID NO: 109), wherein the 5' flanking region and 3' flanking region are each respectively indicated by capital letters, and wherein the gap region is indicated by lower case letters.
  • E48 The gapmer of E34, wherein at least one cytosine is 5-methylcytosine.
  • E50 The gapmer of any of E1 to E15, wherein the nucleobase sequence of said gapmer is CCc ggc ccc ggc ccC GGC (SEQ ID NO: 149), wherein the 5' flanking region and 3' flanking region are each respectively indicated by capital letters, and wherein the gap region is indicated by lower case letters.
  • E51 The gapmer of E37, wherein at least one cytosine is 5-methylcytosine.
  • E52 The gapmer of E37 or E38, wherein cytosines at positions 1 , 2, 3, 9, 15, and 18 are 5-methylcytosine.
  • E53 The gapmer of any of E1 to E15, wherein the nucleobase sequence of said gapmer is CGg ccc egg ccc egg CCC C (SEQ ID NO: 155), wherein the 5' flanking region and 3' flanking region are each respectively indicated by capital letters, and wherein the gap region is indicated by lower case letters.
  • E54 The gapmer of E40, wherein at least one cytosine is 5-methylcytosine.
  • E55 The gapmer of E40 or E41 , wherein cytosines at positions 1 , 7, 13, 16, 17, 18, and 19 are 5-methylcytosine.
  • E56 The gapmer of any of E1 to E15, wherein the nucleobase sequence of said gapmer is CCg gcc ccg gcc ccg GCC C (SEQ ID NO: 158), wherein the 5' flanking region and 3' flanking region are each respectively indicated by capital letters, and wherein the gap region is indicated by lower case letters.
  • E57 The gapmer of E43, wherein at least one cytosine is 5-methylcytosine.
  • E58 The gapmer of E43 or E44, wherein cytosines at positions 1 , 2, 8, 14, 17, 18, and 19 are 5-methylcytosine.
  • E59 The gapmer of any of E1 to E15, wherein the nucleobase sequence of said gapmer is CCC ggc ccc ggc ccc gGC C (SEQ ID NO: 160), wherein the 5' flanking region and 3' flanking region are each respectively indicated by capital letters, and wherein the gap region is indicated by lower case letters.
  • E60 The gapmer of E46, wherein at least one cytosine is 5-methylcytosine.
  • E61 The gapmer of E46 or E47, wherein cytosines at positions 1 , 2, 3, 9, 15, 18, and 19 are 5-methylcytosine.
  • E62 The gapmer of any of E1 to E15, wherein the nucleobase sequence of said gapmer is CCc ggc ccc ggc ccc GGC C (SEQ ID NO: 161), wherein the 5' flanking region and 3' flanking region are each respectively indicated by capital letters, and wherein the gap region is indicated by lower case letters.
  • E63 The gapmer of E49, wherein at least one cytosine is 5-methylcytosine.
  • E64 The gapmer of E49 or E50, wherein cytosines at positions 1 , 2, 3, 9, 15, 18, and 19 are 5-methylcytosine.
  • E65 The gapmer of any of E1 - E51 , covalently conjugated to a nucleotide or non- nucleotide moiety selected from the group consisting of proteins, fatty acid chains, sugar residues, glycoproteins, polymers, or combinations thereof.
  • E66 A composition comprising the gapmer of any of E1 to E65 and a pharmaceutically acceptable carrier.
  • E67 A method of treating a disorder in a subject in need thereof wherein the subject is suffering from a disease or disorder mediated by or associated with repeat expansion, comprising administering to said subject a therapeutically effective amount of the gapmer of any of the preceding claims.
  • E68 The method of E54, wherein said disorder is a neurological disorder.
  • E69 The method of E55, wherein said neurological disorder is a neurodegenerative disease.
  • E70 The method of E56, wherein said neurodegenerative disease is associated with hexanucleotide repeat expansion in the C90RF72 gene.
  • E71 The method of any of E54 to E57, wherein said neurodegenerative disease is selected from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • E72 The method of any of E54 to E58, wherein the gapmer is administered into the central nervous system intrathecally or intraventricularly.
  • E73 Use of the gapmer of any of E1 to E52, or the composition of E53 in the manufacture of a medicament for the treatment of a neurological disorder.
  • E74 The use of E60, wherein said neurological disorder is a neurodegenerative disease.
  • E75 The use of E60, wherein said neurodegenerative disease is associated with hexanucleotide repeat expansion in the C90RF72 gene.
  • E76 The use of E61 or E62, wherein said neurodegenerative disease is selected from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • E77 The use of any of E60 to E63, wherein the gapmer is administered into the central nervous system intrathecally or intraventricularly.
  • E78 A method of inhibiting expression of a C90RF72 transcript containing an expanded hexanucleotide repeat region in a cell, the method comprising contacting a cell containing a target nucleic acid with an effective amount of the gapmer of any of E1 to E52, or the composition of E53.
  • E79 The method of E65, wherein the contacting is in vitro or in vivo.
  • E80 The method of E65 or E66, wherein the C90RF72 transcript is a C90RF72 sense transcript.
  • E81 The method of any of E65 to E67, wherein the C90RF72 transcript is a C90RF72 antisense transcript.
  • E82 The method of any of E65 to E70, wherein the C90RF72 transcript is a pre- mRNA.
  • E83 The method of any of E65 - E69, wherein the gapmer hybridizes with a target sequence present in the expanded hexanucleotide region of the C90RF72 sense transcript or C90RF72 antisense transcript.
  • a gapmer comprising 12 to 30 nucleosides, including a 5' flanking region and a 3' flanking region, each flanking region consisting of 1 to 5 nucleosides, all of which are LNA monomers, and a gap region between the flanking regions consisting of contiguously linked deoxyribonucleosides, wherein said gapmer hybridizes with no more than 6 mismatches to the hexanucleotide repeat region of either C90RF72 sense transcript or C90RF72 antisense transcript.
  • E85 The gapmer of E71 , wherein said gapmer hybridizes with at least 1 mismatch to the hexanucleotide repeat region of a RNA transcript transcribed from either C90RF72 sense transcript or C90RF72 antisense transcript.
  • E86 The gapmer of E71 or E72, wherein said gapmer hybridizes with at least 3 mismatches to the hexanucleotide repeat region of a RNA transcript transcribed from either C90RF72 sense transcript or C90RF72 antisense transcript.
  • E87 The gapmer of any of E71 to E73, wherein said gapmer hybridizes with at least 5 mismatches to the hexanucleotide repeat region of a RNA transcript transcribed from either C90RF72 sense transcript or C90RF72 antisense transcript.
  • E88 The gapmer of any of E71 to E74, wherein all modified nucleosides are LNA monomers.
  • E89 The gapmer of any of E71 to E75, wherein all LNA monomers are beta-D-oxy- LNA monomers.
  • E90 The gapmer of any of E71 to E76, wherein all internucleoside linkages are phosphorothioate.
  • E91 The gapmer of any of E71 to E77, wherein said gapmer comprises at least one modified base.
  • E93 Use of the gapmer of any of E1 to E52, or the composition of E53, for the treatment of a disorder in a subject in need thereof wherein the subject is suffering from a disease or disorder mediated by or associated with repeat expansion.
  • E94 A method of down-regulating the expression of C90RF72 sense transcript containing an expanded hexanucleotide repeat region in a cell, tissue or organism comprising contacting or administering said cell, tissue or organism with an effective amount of one or more of the gapmers of any of E1 to E52, or the compositions of E53.
  • E95 A method of down-regulating the expression of C90RF72 antisense transcript containing an expanded hexanucleotide repeat region in a cell, tissue or organism comprising contacting or administering said cell, tissue or organism with an effective amount of one or more of the gapmers of any of E1 to E52, or the compositions of E53.
  • E96 A method of simultaneously down-regulating the expression of C90RF72 sense transcript containing an expanded hexanucleotide repeat region and C90RF72 antisense transcript containing an expanded hexanucleotide repeat region in a cell, tissue or organism comprising contacting or administering said cell, tissue or organism with an effective amount of one or more of the gapmers of any of E1 to
  • oligomers comprising between 12 to 30 linked nucleosides, at least a 12 nucleobase portion of which is present within a nucleobase sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 176.
  • oligomers are gapmers which, in some embodiments, comprise (in 5' to 3' order), a 5' flanking region consisting of contiguously linked nucleosides, at least one of which is modified, a gap region consisting of contiguously linked deoxyribonucleosides, and a 3' flanking region consisting of contiguously linked nucleosides, at least one of which is modified.
  • the 5' flanking region and 3' flanking region each consist of 1 to 5 nucleosides, all of which are modified.
  • all the modified nucleosides are LNA monomers, including sometimes beta-D-oxy-LNA monomers.
  • all the internucleoside linkages are phosphorothioate.
  • Bases in gapmers can also be modified, for example, by adding a methyl group to one or more cytosines to form 5- methylcytosine.
  • oligomers of the disclosure include those having the following nucleobase sequences: CCc egg ccc egg CC (SEQ ID NO: 12), GCc ccg gec ccg gCC (SEQ ID NO: 13), CCc egg ccc egg ccC C (SEQ ID NO: 14), Ggc ccc ggc ccC (SEQ ID NO:20), CCg gec ccg gcC CC (SEQ ID NO: 103), CCc ggc ccc ggC CC (SEQ ID NO: 106), GGc ccc ggc ccC GG (SEQ ID NO: 109), CCc ggc ccc ggcccC GGC (SEQ ID NO: 149), CGg ccc egg ccc egg CCC C (SEQ ID NO: 155), CCg gee ccg gec cg G
  • the 5' and 3' flanking regions contain LNA monomers, such as LNA nucleosides, indicated by capital letters (e.g., A, T, G, C), whereas the gap regions contain DNA monomers, such as DNA nucleosides, indicated by lower case letters (e.g, a, t, g, c).
  • All the linkages forming the backbone of the exemplary gapmers are phosphorothioate.
  • at least one base in each of the exemplary gapmers is modified.
  • cytosines can be modified by the addition of a methyl group to form 5- methylcytosine.
  • all cytosines in the 5' and 3' flanking regions are 5-methylcytosine, and in the DNA gap region, any cytosine immediately preceding a guanine (e.g., "eg” or "cG”) is 5-methylcytosine, whereas all other cytosines in the gap region remain unmodified.
  • the gapmers of the present invention do not consist of sequences selected from the group consisting of SEQ ID NOs: 189 - 234.
  • Gapmers of the disclosure can be included in compositions also including pharmaceutically acceptable carriers or excipients. Gapmers can also be covalently conjugated to a non-polynucleotide moiety. [009] Gapmers of the disclosure can usefully be employed in methods of preventing or treating disorders, such as neurological disorders in subjects, such as human subjects, by administering to a subject in need of treatment a therapeutically effective amount of a gapmer.
  • the neurological disorder is a neurodegenerative disease, including neurodegenerative diseases associated with hexanudeotide repeat expansion in the C90RF72 gene. Specific examples of such neurodegenerative diseases include amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • Gapmers particularly when included in compositions comprising a pharmaceutically acceptable carrier, can be administered into the central nervous system of a subject intrathecal ⁇ or intraventricularly. Gapmers of the disclosure can additionally be used in the manufacture of a medicament for the treatment of a neurological disorder, such as a neurodegenerative disease, including neurodegenerative diseases associated with hexanudeotide repeat expansion in the C90RF72 gene, for example, ALS and FTD.
  • a neurological disorder such as a neurodegenerative disease, including neurodegenerative diseases associated with hexanudeotide repeat expansion in the C90RF72 gene, for example, ALS and FTD.
  • RNA transcribed from the C90RF72 gene in a cell by contacting the cell in vitro or in vivo with an effective amount of a gapmer of the disclosure.
  • the RNA is transcribed from the minus strand, such as pre-mRNA or mRNA, and in other embodiments, the RNA is transcribed from the plus strand.
  • the gapmer hybridizes with a target sequence present in the hexanudeotide region of RNA transcribed from both the minus and plus strands of the C90RF72 gene and is effective to reduce the amount of one or the other, or both types of RNA so transcribed.
  • the disclosure further provides gapmers comprising 12 to 30 linked nucleosides, including a 5' flanking region and a 3' flanking region, each independently consisting of 1 to 5 nucleosides, all of which are modified, and a gap region consisting of contiguously linked deoxynucleosides positioned between the flanking regions, wherein the gapmer hybridizes with no more than 4 mismatches to the hexanudeotide repeat region of RNA transcripts transcribed from both the minus and plus strands of the C90RF72 gene. In other embodiments, fewer mismatches are allowed, for example, 3 or fewer, 2 or fewer, or not more than 1.
  • all modified nucleosides in the 5' and/or 3' flanking regions are LNA monomers, for example, beta-D-oxy-LNA monomers.
  • all internucleoside linkages are phosphorothioate.
  • at least one base of the gapmer is modified, for example, by the addition of a methyl group, cytosine can be modified to form 5-methylcytosine.
  • FIG. 1 Graph reports data demonstrating effect on C90RF72 pre-mRNA levels in human ND40063 cells of different doses of oligomer numbers 176, 13, 146, and 156 as disclosed in Table 4.
  • FIG. 2. Graph reports data demonstrating effect on levels of sense and antisense RNA produced from C90RF72 in human ND40063 cells of different doses of oligomer numbers 61 , 9, 154, 156, and 158 as disclosed in Table 4.
  • FIG. 3 Graph reports data demonstrating effect on C90RF72 pre-mRNA levels in HEK-293 cells and human ND40063 cells of different doses of oligomer numbers 127, 144, 145, 146, and 147 as disclosed in Table 4.
  • FIG. 4 Nucleic acid sequence of the plus (SEQ ID NO: 185) and minus (SEQ ID NO: 186) strands of an exemplary human C90RF72 gene containing 33 repeats of the hexanucleotide sequence GGGGCC.
  • the sequence shown is based on that of NCBI reference sequence NG_031977.1 , which contains three repeats of the hexanucleotide sequence and one partial repeat.
  • Exon 1 is identified by bold underline font.
  • the hexanucleotide repeat section is identified by bold italic font.
  • Certain oligomers of the disclosure including for example those identified by SEQ ID NOs 31 to 80, target sequences within the region encompassing exon 1 and the sequence up to the beginning of the repeat region.
  • Other oligomers of the disclosure including for example those identified by SEQ ID NOs: 1 to 30 and SEQ ID NOs: 81 to 176, target sequences within the hexanucleotide repeat region.
  • FIG. 5 Graph reports data comparing effect on C90RF72 pre-mRNA levels in ND40063 cells of different doses of oligomer number 109 with 2'-MOE containing oligomers disclosed in Table 5.
  • oligomers capable of down-regulating the expression of C90RF72 by targeting certain complementary nucleobase sequences in the GGGGCC hexanucleotide repeat region of the C9QRF72 gene.
  • oligomers of the disclosure are capable of down-regulating expression of C90RF72 RNA transcribed from the sense (plus) strand of the C90RF72 gene.
  • oligomers are capable of down-regulating the expression of C90RF72 RNA transcribed from the antisense (minus) strand of the C90RF72 gene.
  • the disclosure provides oligomers capable of hybridizing under intracellular conditions to RNA transcribed from the C90RF72 gene which includes the hexanucleotide repeat region of C90RF72.
  • the oligomers target an expanded hexanucleotide repeat region.
  • certain oligomers are incapable of targeting C90RF72 RNA unless it contains two or more hexanucleotide repeats.
  • the RNA is transcribed from the sense strand of the C90RF72 gene, and in other embodiments the RNA is transcribed from the antisense strand of the C90RF72 gene.
  • oligomers of the disclosure down-regulate RNA expression from the C90RF72 gene, thereby reducing the incidence of intranuclear RNA foci or intracellular aggregates of protein produced from transcripts of the C90RF72 gene containing hexanucleotide repeats.
  • the oligomers of the disclosure are composed of covalently linked monomers.
  • nucleosides include both nucleosides and deoxynucleosides (collectively, “nucleosides”) that occur naturally in nucleic acids and that contain neither modified sugars nor modified nucleobases, i.e., compounds in which a ribose sugar or deoxyribose sugar is covalently bonded to a naturally-occurring, unmodified nucleobase (sometimes simply called a base) moiety (i.e., the purine and pyrimidine heterocycles adenine, guanine, cytosine, thymine or uracil, abbreviated A, G, C, T, and U, respectively) and “nucleoside analogues,” which are nucleosides that either do occur naturally in nucleic acids or do not occur naturally in nucleic acids, wherein either the sugar moiety is other than a ribose or a deoxyribose sugar (such as bicyclic sugars or 2' modified sugars
  • RNA monomer is a nucleoside containing a ribose sugar and an unmodified nucleobase.
  • a “DNA monomer” is a nucleoside containing a deoxyribose sugar and an unmodified nucleobase.
  • a “Locked Nucleic Acid monomer,” “locked monomer,” or “LNA monomer” is a nucleoside analogue having a bicyclic sugar, as further described herein.
  • a "modified base”, as used herein, refers to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil.
  • 5-methylcytosine refers to a cytosine modified with a methyl group attached to the 5' position.
  • a 5-methylcytosine is a modified base.
  • a "2'-0-methoxyethyl group' refers to an O-methoxy-ethyl modification of the 2' position of a furanosyl ring.
  • a 2'-0-methoxyethyl modified sugar is a modified sugar.
  • a "2'-MOE nucleoside” refers to a nucleoside comprising a 2'-0-methoxyethyl group.
  • nucleoside analogue and “corresponding nucleoside” indicate that the base moiety in the nucleoside analogue and the base moiety in the nucleoside are identical.
  • nucleoside analogue contains, for example, a modified sugar linked to an adenine base moiety.
  • oligomer refers to a molecule formed by covalent linkage of two or more contiguous monomers by, for example, a phosphate group (forming a phosphodiester linkage between adjacent nucleosides) or a phosphorothioate group (forming a phosphorothioate linkage between adjacent nucleosides).
  • oligomers of the disclosure comprise or consist of 8-50 monomers, such as 10-30 monomers.
  • an oligomer comprises nucleosides, or nucleoside analogues, or mixtures thereof as referred to herein.
  • the terms "LNA oligomer” or “LNA oligonucleotide” refer to an oligonucleotide containing one or more LNA monomers.
  • oligomers according to the disclosure comprise at least one nucleoside analogue monomer, such as an LNA monomer, or other nucleoside analogue monomer.
  • Nucleoside analogues optionally included within oligomers may function similarly to corresponding nucleosides, or may have distinct functions. Oligomers in which some or all of the monomers are nucleoside analogues may have improved properties compared to oligomers lacking such analogues making them function better as drugs. Such improved properties may include the ability to cross the blood-brain-barrier, penetrate cell membranes, resistance to extracellular and/or intracellular nucleases and high affinity and specificity for the nucleic acid target. In some embodiments, LNA monomers possess several of the above-mentioned improved properties.
  • Nucleoside analogues may also be "silent” or “equivalent” in function to the corresponding unmodified nucleoside so that they have no known effect on the way the oligomer functions to inhibit target gene expression and/or its resistance to nucleases. Such "equivalent" nucleoside analogues may nevertheless be useful in that they are easier or less costly to manufacture, be more stable under storage or manufacturing conditions, or serve as an attachment point for a tag or label.
  • the term "at least one” means integers equal to or larger than 1 , such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 and so forth.
  • the term “at least one” encompasses the terms “at least two” and “at least three” and “at least four,” etc.
  • the term “at least two” comprises the terms “at least three,” “at least four,” etc.
  • an oligomer consists of 8-50 contiguously linked monomers, such as 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguously linked monomers. In other embodiments, an oligomer consists of 10-25 monomers, 10-16 monomers, or 12-16 monomers.
  • an oligomer of the disclosure does not comprise RNA monomers (e.g., RNA nucleosides or nucleotides or other RNA monomers).
  • an oligomer comprises DNA monomers (e.g., DNA nucleosides or nucleotides or other DNA monomers).
  • region means a certain number of contiguously linked monomers within the overall oligomer sequence that is less than the overall length of the oligomer, defined as the total number of monomers in the oligomer.
  • oligomers are single-stranded, molecules that exclude any substantially self-complementary regions, thereby avoiding or reducing the likelihood that intrastrand base pairing can occur, or that an internal duplex can form.
  • oligomers are not substantially double-stranded, or are not siRNA.
  • the oligomers of the disclosure consist of a contiguously linked stretch of monomers the base sequences of which, and corresponding sequence identification numbers, are disclosed in one or more Tables set forth herein.
  • nucleic acid and polynucleotide are used interchangeably herein and are defined as a molecule formed by covalent linkage of two or more monomers as that term is described elsewhere herein.
  • a nucleic acid can be any length, starting with two monomers.
  • nucleic acid and polynucleotide are generic to oligomer, which has structure and length described elsewhere herein.
  • nucleic acid and polynucleotide include, but are not limited to, single-stranded, double-stranded, partially double-stranded, self-complementary and circular nucleic acids and polynucleotides.
  • the human C90RF72 gene (SEQ ID NO: 187) is located on chromosome 9 at 9p21 .2.
  • the reference sequence for the gene has GenBank Accession No. NG_031977.1 , which is incorporated by reference.
  • exon 1 corresponds to bases 5001 -5158
  • exon 2 corresponds to bases 1 1703-12190
  • exon 3 corresponds to bases 13277-13336
  • exon 4 corresponds to bases 16391 -16486
  • exon 5 corresponds to bases 17218-17282
  • exon 6 corresponds to bases 18568-18640
  • exon 7 corresponds to bases 20260-20376
  • exon 8 corresponds to bases 22071 -22306
  • exon 9 corresponds to bases 28160-28217
  • exon 10 corresponds to bases 30201 -30310
  • exon 1 1 corresponds to bases 30445-32322.
  • Exon 1 is non-coding
  • exons 2-1 1 have the potential to encode protein depending on the result of alternative mRNA splicing.
  • Transcript variant 1 is predicted to encode a 222 amino acid protein encoded by C90RF72 exons 2-5 and is called "protein isoform b.”
  • the nucleic acid sequence of transcript variant 1 is set forth as GenBank Accession No. NM_145005.6 (SEQ ID NO:188) and the amino acid sequence of isoform b is set forth as GenBank Accession No. NP_659442.2. Each of these sequences is incorporated by reference herein.
  • transcript variants 2 and 3 are predicted to encode the same 481 amino acid protein encoded by C90RF72 exons 2 through 1 1 which is called "protein isoform a.”
  • the nucleic acid sequence of transcript variant 2 is set forth as GenBank Accession No. NM_018325.3
  • the nucleic acid sequence of transcript variant 3 is set forth as GenBank Accession No. NM_001256054.1
  • the amino acid sequence of isoform a is set forth as GenBank Accession No. NP_060795.1 .
  • Each of these sequences is incorporated by reference herein.
  • the hexanucleotide (GGGGCC) repeat region if present, is positioned in the intron located between exons 1 a and 1 b, usually closer to the first exon.
  • a limited number of repeats of the GGGGCC sequence have been found to be non-pathogenic in humans.
  • healthy individuals have an average of two repeats of the GGGGCC sequence, but can possess as many as about 20, whereas expansion to about 30 or more GGGGCC repeats is statistically correlated with developing the neurological diseases ALS or FTD.
  • the typical number of repeats in affected individuals can be many fold higher, however, such as including up to 700 to 1600 hexanucleotide repeats, or more. DeJesus-Hernandez, et al., Neuron 72:245-256 (201 1 ); Renton, et al., Neuron 72:257-268 (201 1).
  • FIG. 4 provides the nucleic acid sequence of the plus strand of an exemplary human C90RF72 gene containing 33 repeats of the hexanucleotide sequence GGGGCC.
  • the sequence shown is based on that of NCBI reference sequence NG_031977.1 , which contains three repeats of the hexanucleotide sequence and one partial repeat (GGGGC).
  • Exon 1 is identified by bold underline font, whereas the hexanucleotide repeat section is identified by bold italic font.
  • the sequence of FIG. 4, however, is merely exemplary and should not be construed as limiting.
  • variants or alleles of the C90RF72 gene exist in human populations, for example, with fewer or a greater number of hexanucleotide repeats.
  • Partial repeats and/or other sequence variants such as single nucleotide polymorphisms (SNPs), or other variants, may also be present adjacent to or outside the hexanucleotide repeat region.
  • SNPs single nucleotide polymorphisms
  • target nucleic acid refers to the C90RF72 gene (and naturally occurring variants thereof) as it exists in the genome or as isolated therefrom (such as genomic fragments), including exons and introns (including the hexanucleotide repeat region), genetic control regions (such as promoters and enhancers), splice junctions, and 5' and 3' untranslated regions (UTR).
  • Target nucleic acid also includes any RNA transcribed from the plus strand or minus strand of the C90RF72 gene.
  • strand of the C90RF72 gene refers to the DNA strand of the C90RF72 gene comprising the same nucleobase sequence (except for thymine in DNA which is replaced by uracil in RNA) as the pre-mRNA which, after splicing, generates mRNA translatable to any of the known C90RF72 protein isoforms.
  • the "minus strand” of the C90RF72 gene refers to the DNA strand of the C90RF72 gene comprising the nucleobase sequence that is transcribed by cellular RNA polymerase to synthesize the C90RF72 pre-mRNA described immediately above.
  • the minus strand of the C90RF72 gene is complementary to the plus strand and vice versa.
  • RNA transcripts within the term target nucleic acid include C90RF72 pre-mRNA and mRNA encoding the C90RF72 protein, including the three known mRNA variants described above, and any new mRNA variants that may yet be discovered. Additionally encompassed by the term target nucleic acid is cDNA prepared from such mRNA.
  • the present invention contemplates gapmers, uses and compositions thereof, wherein the gapmer is capable of preferentially inhibiting expression of C90RF72 sense transcript containing an expanded hexanucleotide repeat region when compared with inhibiting expression of a wild-type C90RF72 sense transcript.
  • a "C90RF72 sense transcript” refers to RNA that is transcribed from the sense strand of the C90RF72 gene and which also contains the hexanucleotide repeat region found between exons 1 a and 1 b.
  • the C90RF72 sense transcript will have areas of substantial sequence identity to the C90RF72 mRNA sequence, for example, SEQ ID NO:188, but will additionally contain intronic sequence, particularly at least the aforementioned hexanucleotide repeat region.
  • a transcript is said to have an "expanded hexanucleotide repeat region" when it has a pathogenic number of hexanucleotide repeats [for the sense transcript, (GGGGCC)n, and for the antisense transcript, (GGCCCC) n ].
  • this number is significantly higher than the number of repeats found in the C90RF72 gene in a normal subject (i.e., a human subject that is not suffering from familiar ALS or FTD), the latter of which generally has 30 or fewer repeats.
  • Exanded hexanucleotide repeat regions of the C90RF72 gene can be detected using means known in the art.
  • an expanded hexanucleotide repeat region is a transcript which has more than 30 hexanucleotide repeats, for example, at least 40 repeats, at least 50 repeats, at least 60 repeats, at least 75 repeats, at least 100 repeats, at least 125, repeats, at least 150 repeats, at least 200 repeats, at least 250 repeats, at least 300 repeats, or more hexanucleotide repeats.
  • the gapmers of the present invention also are capable of inhibiting expression of a C90RF72 antisense transcript.
  • a "C90RF72 antisense transcript” refers to RNA that is transcribed from the antisense strand of the C90RF72 gene and which also contains the hexanucleotide repeat region found between exons 1 a and 1 b.
  • the C90RF72 antisense transcript will have areas of substantial sequence complementarity to the C90RF72 mRNA sequence, for example, SEQ ID NO:188, but additionally contain intron sequences, particularly the hexanucleotide repeat region (GGCCCC) n .
  • Target nucleic acid also includes RNA transcripts transcribed from the plus (sense) or minus (antisense) strand of the C90RF72 gene that are not spliced into mRNA encoding C90RF72 protein.
  • RNA transcripts include those present within RNA foci in cells (e.g., brain cells, fibroblasts, white blood cells or other types of cells) from patients with C90RF72 repeat expansion, as well as RNA transcripts translatable into proteins containing dipeptide repeats encoded by the hexanucleotide repeats, whether translated by repeat- associated non-ATG (RAN) translation or some other mechanism.
  • proteins containing dipeptide repeats are translated from plus strand RNA transcribed from the antisense strand where the dipeptide repeats are Gly-Ala (e.g., GA or AG), Gly-Pro (e.g., GP or PG) or Gly-Arg (e.g., GR or RG).
  • proteins containing dipeptide repeats are encoded by minus strand RNA transcribed from the sense strand where the dipeptide repeats are Gly-Pro (e.g., GP or PG), Ala-Pro (e.g., AP or PA) or Pro-Arg (e.g., PR or RP).
  • target nucleic acid any naturally occurring variants of the plus or minus DNA strands of the C90RF72 gene and RNA transcribed therefrom, including for example pre-mRNA and mRNA.
  • naturally occurring variant refers to any allelic variants of the C90RF72 gene existing naturally within the human population , and RNA, including pre-mRNA and mRNA, transcribed from such allelic gene variants, and proteins encoded by such allelic gene variants.
  • the term includes naturally occurring forms of the protein (e.g., isoform a or isoform b) and proteins having undergone co-translational or post- translational processing, such as signal peptide cleavage, proteolytic cleavage, or glycosylation.
  • Naturally occurring variants of the C90RF72 protein also includes proteins comprising repeating dipeptide sequences resulting from repeat associated non-ATG (RAN) translation from RNA transcribed from the C90RF72 gene, as described more fully in Mori, et al., Science 339:1335-1338 (2013), and Ash, et al., Neuron 77, 639-646 (2013).
  • Naturally occurring variants include alleles of the C90RF72 gene containing a pathogenic number of GGGGCC hexanucleotide repeats.
  • Naturally occurring variants also includes RNA transcribed from the antisense strand comprising such hexanucleotide repeats (including pre-mRNA and mRNA), as well as RNA transcribed from the sense strand comprising the reverse complement of the hexanucleotide repeats (i.e., GGCCCC).
  • the nucleic acid sequence of the expanded region is provided by the formula (GGGGCC)n, or (GGCCCC)n wherein GGGGCC or GGCCCC is a single hexanucleotide repeat unit, and wherein "n" is a number that varies from 0 to 5000 or more.
  • n can be 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more, including any integer between or among the previously specified values.
  • the nucleobase sequence of a hexanucleotide repeat unit depends on which strand is being read.
  • the repeated hexanucleotide sequence is 5'-GGGGCC-3' and the sequence would be the same in any RNA transcribed from the complementary minus strand.
  • the repeated hexanucleotide sequence is 5'- GGCCCC-3' (i.e., the reverse complement) and the sequence would be the same in any RNA transcribed from the complementary plus strand.
  • RNA transcribed from the "minus" or antisense strand of C90RF72 has not been identified, it is known to contain the hexanucleotide repeat [albeit in the complementary sequence (i.e., GGCCCC)], as both sense and antisense transcripts containing the intron 1 sequence (where the GGGGCC repeat is located) were found to be strongly increased in C90RF72 patients (Mori et al., ibid), and nuclear RNA foci containing GGCCCC-containing repeats have been identified in fibroblasts from C90RF72 patients (Lagier-Tourenne et al., ibid)
  • the nucleic acid sequence of the repeat region when viewed from the perspective of the plus strand, is provided by the formula (GGGGCC) n , wherein GGGGCC is a single hexanucleotide repeat unit and wherein "n” is an integer that varies from 0 to 5000 or more.
  • the nucleic acid sequence of the expanded region when viewed from the perspective of the minus strand, is provided by the formula (GGCCCC) n , wherein GGCCCC is a single hexanucleotide repeat unit, and wherein "n” is an integer that varies from 0 to 5000 or more.
  • n in the formulae above can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more, including any integer between or among the previously specified values.
  • the hexanucleotide repeat region can be flanked on either the 5' or 3' end, or both the 5' and 3' ends, by a single partial repeat.
  • a partial repeat positioned at the 5' end of the repeat region can have the sequence GGGCC, GGCC, GCC, CC, and C
  • a partial repeat positioned at the 3' end of the repeat region can have the sequence GGGGC, GGGG, GGG, GG, and G.
  • a partial repeat positioned at the 5' end of the repeat region can have the sequence, GCCCC, CCCC, CCC, CC, or C
  • a partial repeat positioned at the 3' end of the repeat region can have the sequence GGCCC, GGCC, GGC, GG, or G.
  • oligomers of the disclosure can hybridize with a nucleobase sequence present in the plus strand of the C90RF72 gene or corresponding RNA transcript. In other embodiments, oligomers of the disclosure can hybridize with a nucleobase sequence present in the minus strand of the C90RF72 gene or corresponding RNA transcript. And in yet other embodiments, oligomers of the disclosure can hybridize with a nucleobase sequence present in both the plus strand and minus strand of the C90RF72 gene or corresponding RNA transcript. In the latter embodiments, the sequences in the plus and minus strands targeted by the oligomers need not be identical.
  • Oligomers of the disclosure hybridize to a region in the target nucleic acid (the "target region") having a sequence of nucleobases complementary or partially complementary to those in the oligomer. Accordingly, the sequence of nucleobases in the oligomer is identical or substantially identical to the reverse complement sequence of the region to which the oligomer hybridizes. If the sequence of the oligomer and the reverse complement sequence of the target region are identical, then the oligomer is "complementary" to the target region. If, however, the sequence of the oligomer and the reverse complement sequence of the target region are similar but not identical, then the oligomer is "partially complementary" to the target region.
  • hybridization typically occurs by Watson-Crick base pairing, although Hoogsteen hydrogen bonding and reversed Hoogsteen hydrogen bonding may also be possible.
  • the mechanism by which hybridization may occur in any particular case, however, is not intended to be limiting in any way.
  • an oligomer's target region is the nucleobase sequence in a target nucleic acid having the highest percentage complementarity to the oligomer.
  • the percentage complementarity between the oligomer and its target region can be calculated by counting the number of identical bases between the oligomer sequence and the reverse complement sequence of the target region, dividing by the total length of the oligomer, and then multiplying the quotient by 100.
  • an oligomer may be able to hybridize to a target region even when the percentage complementarity is less than 100% by virtue of the existence of one or more mismatches between the complementary sequences of the oligomer and target region.
  • mismatch refers to nonidentity of a nucleobase between two sequences under comparison, for example, as between the nucleobase sequence of an oligomer and the reverse complement of the target region to which it hybridizes. Mismatches can consist of a single nucleobase difference or a plurality of nucleobase differences. Typically, mismatches are considered in the context of larger sequences that are otherwise substantially similar.
  • Identifying an oligomer's target region or regions in a target nucleic acid can efficiently be carried out using computer bioinformatics software implementing a sequence alignment algorithm.
  • a variety of such programs and algorithms are known in the art, which can also calculate percentage complementarity (also called percentage identity or percentage homology).
  • percentage complementarity also called percentage identity or percentage homology.
  • a non-limiting example is software implementing the Smith-Waterman algorithm available from the European Bioinformatics Institute (http://www.ebi.ac.uk/Tools/emboss/) or from the BLAST software suite available from National Institute of Medicine (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
  • oligomers of the disclosure are capable of reducing the amount of RNA that is transcribed from the C90RF72 gene, or otherwise inhibiting the expression of such RNA.
  • the RNA is a C90RF72 sense transcript.
  • the RNA is a C90RF72 antisense transcript.
  • the oligomers of the present invention are capable of inhibiting both a C90RF72 sense transcript and C90RF72 antisense transcript.
  • oligomers of the disclosure can reduce the average size and/or number of RNA foci containing such transcripts in the cells of patients with ALS, FTD, or other disease or disorder attributed to hexanucleotide repeat expansion in the C90RF72 gene. Oligomers of the disclosure can also reduce the amount of pre-mRNA and mRNA encoding C90RF72 protein in its various isoforms.
  • the gene and RNA contain hexanucleotide repeats as defined herein.
  • the gapmers of the present invention are capable of preferentially inhibiting expression of a C90RF72 sense transcript containing an expanded hexanucleotide repeat when compared with inhibiting expression of a wild-type C90RF72 sense transcript.
  • the oligomers of the present invention with a half-maximal inhibitory concentration (IC 50 ) for a C90RF72 sense transcript containing an expanded hexanucleotide repeat that is at least 50% less than the IC50 for a wild-type C90RF72 sense transcript (i.e., a transcript containing, for example 20 or fewer hexanucleotide repeats, for example, 15 or fewer, 10 or fewer, 8 or fewer, or less, hexanucleotide repeats).
  • IC 50 half-maximal inhibitory concentration
  • the gapmer of the present invention can have an IC 50 for the C90RF72 sense transcript containing an expanded hexanucleotide repeat that is 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 3% or less, 2% or less, 1 % or less than the IC50 for a wild-type C90RF72 sense transcript (i.e., not containing an expanded hexanucleotide repeat).
  • Methods for determining the IC50 of an oligomer for inhibition of expression of a C90RF transcript are described hereinbelow in the Examples section.
  • the IC 50 is determined in vitro using cells, for example, fibroblasts or kidney cells, that contain either a wild-type human C90RF72 or one containing expanded hexanucleotide repeats (for example, fibroblasts such as ND40063 cells, which were isolated from patients with familial FTD/ALS and confirmed to have expanded hexanucleotide repeats).
  • Oligomers are provided to the cells at defined concentrations and delivered into the cells using any number of methods known in the art. For example, oligomers can be introduced by gymnotic delivery (see Example 1 ; see also Soifer, H.S., et. al., (2012) Methods Mol Biol.
  • RNA is extracted and the relative amounts of the C90RF72 sense transcript and/or C90RF72 antisense transcript are measured and compared to an internal control using methods known in the art (e.g., RT-PCR, RNA-SEQ. etc.) using primers designed to detect the respective transcripts (SEQ ID Nos: 179-184; see Example 1 ).
  • the IC50 is determined by plotting the relative abundance of the transcript against the concentration, and establishing the concentration at which 50% of maximal inhibition occurs.
  • the ability of an oligomer, including the gapmers of the present invention to preferentially inhibit expression of a C90RF72 sense transcript with an expanded hexanucleotide repeat region when compared with a wild-type C90RF72 sense transcript is measured using ND40063 and wild-type human fibroblast cells, by gymnotic delivery of oligomers, and detecting transcript levels by RT-PCR 72 hrs after delivery, using the primers and conditions described in the Examples section. Methods described above can also be used to determine the ability of an oligomer, including the gapmers of the present invention, to inhibit expression of a C90RF72 antisense transcript.
  • oligomers of the disclosure can indirectly reduce the amount of protein, if any, encoded by the transcribed RNA.
  • the protein contains dipeptide repeats encoded by the hexanucleotide repeat region in the C90RF72 gene, whether from the plus or minus strand, or from both strands.
  • the protein is C90RF72 protein in its various isoforms.
  • oligomers of the disclosure can reduce the average size and/or number of intracellular aggregates comprising such proteins accumulating in the cells of patients with ALS, FTD, or other disease or disorder attributed to hexanucleotide repeat expansion in the C90RF72 gene.
  • oligomers of the disclosure function to reduce the amount of RNA that is transcribed from the C90RF72 gene by hybridizing with a target region in the C90RF72 gene (in the plus and/or minus strand) and inhibiting transcription of the strand containing the target region into RNA.
  • oligomers of the disclosure function to reduce the amount of RNA that is transcribed from the C90RF72 gene by hybridizing with a target region in RNA that has already been transcribed from the plus and/or minus strand of the C90RF72 gene. Examples of such RNA include unspliced RNA, pre- mRNA or mRNA.
  • oligomers of the disclosure promote the destruction of transcribed RNA by endogenous nucleases, and/or inhibit or prevent translation of the RNA into protein.
  • Other mechanisms of reducing the amount of RNA that is transcribed from the C90RF72 gene by the oligomers of the disclosure are possible and the mechanism of action is not intended to limit the scope of the invention in any respect.
  • oligomers of the disclosure reduce the amount RNA that has already been transcribed from the minus strand of the C90RF72 gene (e.g., unspliced RNA, pre-mRNA or mRNA) in a cell at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% or more as compared to the amount of C90RF72 RNA that would be present in the absence of the oligomers.
  • RNA that has already been transcribed from the minus strand of the C90RF72 gene e.g., unspliced RNA, pre-mRNA or mRNA
  • oligomers of the disclosure reduce the amount of RNA transcription from the minus strand of the C90RF72 gene in a cell at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% or more as compared to the amount of transcription in the absence of the oligomers. In other embodiments, oligomers of the disclosure reduce the amount C90RF72 protein in a cell at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% or more as compared to the amount of C90RF72 protein that would be present in the absence of the oligomers.
  • oligomers of the disclosure reduce the amount RNA that has already been transcribed from the plus strand of the C90RF72 gene in a cell (e.g., unspliced RNA, pre-mRNA or mRNA) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% or more as compared to the amount of C90RF72 RNA that would otherwise be present in the cell the absence of the oligomers.
  • a cell e.g., unspliced RNA, pre-mRNA or mRNA
  • oligomers of the disclosure reduce the amount of RNA transcription from the plus strand of the C90RF72 gene in a cell at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% or more as compared to the amount of transcription in the absence of the oligomers.
  • C90RF72 RNA in cells or the amount of C90RF72 RNA transcribed or translated into C90RF72 protein
  • the amount of C90RF72 RNA can be detected using Northern blots, or more sensitive techniques such as quantitative RT-PCR and related techniques.
  • the amount of C90RF72 protein can be measured using Western blots, ELISA or RIA using an antibody that specifically binds C90RF72 protein.
  • Other techniques that can be used are also well-known in the art.
  • the efficiency with which a particular oligomer is effective to reduce or inhibit the amount of C90RF72 RNA in a cell depends on a variety of factors, such as its length, melting temperature and ability to recruit RNase H, and possibly other factors. Irrespective of the underlying mechanism(s), the inhibitory efficiency of an oligomer may be determined empirically and expressed as the 50% inhibitory concentration, abbreviated IC 50 .
  • the IC 50 value of an oligomer can be determined by treating test cells expressing the C90RF72 gene, for example certain human cells in vitro, with a range of concentrations of the oligomer and then determine what concentration would reduce the amount of C90RF72 RNA, transcription and/or translation by 50% compared to untreated control cells. Methods for performing such experiments are known in the art.
  • oligomers can be introduced into test cells using a transfection reagent such as lipofectamine. Alternatively, no transfection reagent is used, but rather the oligomers are simply added to the cells in a physiologically compatible fluid, such as PBS or growth medium. An example of this approach is called gymnosis.
  • test cells are fibroblasts or other cells isolated from human patients diagnosed with familial ALS or familial FTD caused by hexanucleotide repeat expansion.
  • oligomers of the disclosure have an IC 50 value in the micromolar range, e.g., 1 -10 uM, 5-20 uM, 10-20 uM, 15-25 uM, 20-30 uM, 25-35 uM, 30-40 uM, 35-45 uM, 40-50 uM, 45-55 uM, 50-60 uM, 55-65 uM, 60-70 uM, 65-75 uM, 70-80 uM, 75-85 uM, 80-90 uM, 85-95 uM, 90-100 uM, 100-150 uM, 150-200 uM, 200-300 uM, 300-400 uM, 400-500 uM or more.
  • IC 50 value in the micromolar range e.g., 1 -10 uM, 5-20 uM, 10-20 uM, 15-25 uM, 20-30 uM, 25-35 uM, 30-40 uM, 35-45
  • oligomers of the disclosure have an IC 50 value in the nanomolar range, e.g., 1 -10 nM, 5-20 nM, 10-20 nM, 15-25 nM, 20-30 nM, 25-35 nM, 30-40 nM, 35-45 nM, 40-50 nM, 45-55 nM, 50-60 nM, 55-65 nM, 60-70 nM, 65-75 nM, 70-80 nM, 75- 85 nM, 80-90 nM, 85-95 nM, 90-100 nM, 100-150 nM, 150-200 nM, 200-300 nM, 300-400 nM, 400-500 nM or more.
  • IC 50 value in the nanomolar range e.g., 1 -10 nM, 5-20 nM, 10-20 nM, 15-25 nM, 20-30 nM, 25-35 nM, 30-40 nM, 35-45
  • oligomers of the disclosure have an IC 50 value in the picomolar range, e.g., 30-40 pM, 35-45 pM, 40-50 pM, 45-55 pM, 50-60 pM, 55-65 pM, 60-70 pM, 65-75 pM, 70-80 pM, 75-85 pM, 80-90 pM, 85-95 pM, 90-100 pM, 100-150 pM, 150-200 pM, 200-300 pM, 300-400 pM, 400-500 pM, 500-600 pM, 600-700 pM, 700-800 pM, 800-900 pM, or 900-1000 pM.
  • IC 50 value in the picomolar range e.g., 30-40 pM, 35-45 pM, 40-50 pM, 45-55 pM, 50-60 pM, 55-65 pM, 60-70 pM, 65-75 p
  • IC50 values can vary depending on the ability of the oligomers to enter the cell.
  • IC50 values are determined in vitro in cells after oligomer delivery using a method selected from gymnotic delivery, using transfection reagents containing cationic lipid or lipid derivative, or electroporation.
  • IC50 values can be determined after gymnotic delivery of the oligomers.
  • oligomers of the disclosure have an IC 50 value in the submicromolar range, i.e., less than 1 uM, less than 500 nM, less than 250 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM or less than 2 nM. In some other embodiments oligomers of the disclosure have an IC 50 value in the subnanomolar range, i.e., less than 1 nM, less than 500 pM, less than 250 pM, less than 100 pM, less than 50 pM, or less than 10 pM.
  • the disclosure additionally provides methods of inhibiting expression from the C90RF72 gene.
  • the disclosure provides a method for inhibiting C90RF72 expression by contacting a cell expressing the C90RF72 gene with an amount of an oligomer of the disclosure effective to inhibit expression of the C90RF72 gene.
  • the oligomer hybridizes to a target region on the plus and/or minus strand of the C90RF72 gene, or RNA produced from the plus and/or minus strand.
  • oligomers of the disclosure capable of hybridizing to target regions in the human C90RF72 gene (plus and/or minus strand), or RNA produced therefrom, are disclosed in TABLE 4 in the Examples section.
  • the disclosure provides oligomers having a nucleobase sequence that differs by 1 or more, 2 or more, 3 or more, or 4 or more bases (i.e., mismatches relative to complementary target regions) from those oligomers listed in TABLE 4, yet are still capable of hybridizing to the C90RF72 gene or RNA produced therefrom and thereby inhibit production or stability of C90RF72 RNA.
  • the destabilizing effect of mismatches on duplex stability may, for example, be compensated for by increased oligomer length and/or increased number of nucleoside analogues, such as LNA monomers, present within the oligomer.
  • the number of mismatches is 4 or less, 3 or less, 2 or less, or only 1 .
  • the disclosure includes oligomers at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the oligomers specifically exemplified by in Table 4 having SEQ ID NO:1 to SEQ ID NO:176, wherein said similar oligomers are still capable of hybridizing to the C90RF72 gene or RNA produced therefrom and thereby inhibit production or stability of C90RF72 RNA.
  • the oligomer comprises additional monomers at the 5' end only, the 3' end only or at both 5' and 3' ends that are non-complementary to the sequence of the target region.
  • as many as 5 additional monomers e.g., 1 , 2, 3, 4 or 5 additional monomers, can be placed at either the 5' end, the 3' end, or both 5' and 3' ends.
  • an oligomer can comprise a region that is complementary to a target region in the C90RF72 gene, as well as non-complementary flanking region at the 5' and/or 3' end of the oligomer.
  • the flanking non-complementary monomers are DNA or RNA monomers, for example, nucleotides or nucleosides.
  • Oligomers of the disclosure may be synthesized using any technique familiar to those of ordinary skill, for example, the technique of solid phase synthesis.
  • the oligomer comprises or consists of 10-50, 1 1 -48, 12-46, 13-44, 14-42, 15-40, 16-38, 17-36, 1 8-34, 1 9-32, 20-30, 21 -28, 22-26 contiguous monomers.
  • oligomers comprise or consist of 10-16, 10-18, 10-22, 10- 24, 10-25, 12-14, 12-16, 12-18, 12-24, 12-25, 1 3-17, 14-16, or 14-18 contiguous monomers.
  • the oligomer comprises or consists of 8, 9, 10, 1 1 , 12, 1 3, 14, 15, 16, 17, 1 8, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 contiguous monomers.
  • the oligomer consists of no more than 22, 21 , 20, 19, 18, 17, 16, 1 5 or fewer contiguous monomers.
  • the oligomers of the present invention, including gapmers described herein consist of between 12 and 20 monomers.
  • the modified nucleoside analogue contains a modified base, such as a base selected from the group consisting of 5- methylcytosine (which can be abbreviated m5C, or C5Me) , isocytosine, pseudoisocytosine, 5- bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2- chloro-6-aminopurine, xanthine, hypoxanthine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6- aminopurine.
  • 5- methylcytosine which can be abbreviated m5C, or C5Me
  • isocytosine, pseudoisocytosine such as a base selected from the group consisting of 5-
  • At least one of the monomers of the oligomer is a nucleoside analogue that contains a modified sugar.
  • the linkage between at least two contiguous monomers of the oligomer is other than a phosphodiester bond.
  • oligomers include at least one monomer that has a modified base, at least one monomer having a modified sugar (which may be the same monomer having the modified base) , and at least one inter-monomer linkage that is other than a phosphodiester bond .
  • nucleoside analogues useful in the oligomers described herein are described by e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development; 2000, 3(2), 293-213, and in Scheme 1 (in which some nucleoside analogues are shown in the form of nucleotides):
  • the oligomer may thus comprise or consist of a sequence of linked nucleosides, including for example DNA or RNA, or a combination of nucleosides and one or more nucleoside analogues.
  • nucleoside analogues suitably enhance the affinity of the oligomer for the target region of the target nucleic acid.
  • nucleoside analogue comprises a sugar moiety modified to provide a 2'-substituent group, such as 2'-0-alkyl-ribose sugars, 2'-amino-deoxyribose sugars, and 2'-fluoro-deoxyribose sugars.
  • the nucleoside analogue comprises a sugar in which a bridged structure, creating a bicyclic sugar (LNA), is present, which can enhance binding affinity and may also provide some increased nuclease resistance.
  • the LNA monomer is selected from oxy-LNA (such as beta-D-oxy-LNA, and alpha-L-oxy-LNA), and/or amino-LNA (such as beta-D-amino-LNA and alpha-L-amino-LNA) and/or thio-LNA (such as beta-D-thio-LNA and alpha-L-thio-LNA) and/or ENA (such as beta-D-ENA and alpha-L-ENA).
  • the LNA monomers are beta-D-oxy-LNA. LNA monomers are further described, below.
  • affinity-enhancing nucleoside analogues in the oligomer can allow the size of the oligomer to be reduced, and may also reduce the upper limit to the size of the oligomer before non-specific or aberrant binding takes place.
  • the oligomer comprises at least 2 nucleoside analogues. In some embodiments, the oligomer comprises from 3-8 nucleoside analogues, e.g. 6 or 7 nucleoside analogues. In preferred embodiments, at least one of the nucleoside analogues is a locked nucleic acid (LNA) monomer; for example at least 3 or at least 4, or at least 5, or at least 6, or at least 7, or 8, of the nucleoside analogues are LNA monomers. In some embodiments all the nucleoside analogues are LNA monomers.
  • LNA locked nucleic acid
  • the oligomers comprise a corresponding nucleoside analogue, such as a corresponding LNA monomer or other corresponding nucleoside analogue, which raise the duplex stability, e.g., melting temperature (Tm), of the oligomer/target region duplex (i.e., affinity enhancing nucleoside analogues).
  • a corresponding nucleoside analogue such as a corresponding LNA monomer or other corresponding nucleoside analogue, which raise the duplex stability, e.g., melting temperature (Tm), of the oligomer/target region duplex (i.e., affinity enhancing nucleoside analogues).
  • any mismatches (that is, noncomplementarities) between the base sequence of the oligomer and the base sequence of the target region are located other than in the regions of the oligomer that contain affinity-enhancing nucleoside analogues (e.g., regions A or C), such as within region B as referred to herein below, and/or within region D as referred to herein below, and/or in regions of the oligomer containing only nucleosides, and/or in regions which are 5' or 3' to the region of the oligomer that is complementary to the target region.
  • affinity-enhancing nucleoside analogues e.g., regions A or C
  • nucleoside analogues present within the oligomer of the disclosure are independently selected from, for example: monomers containing 2'-0-alkyl-ribose sugars, monomers containing 2'-amino- deoxyribose sugars, monomers containing 2'-fluoro-deoxyribose sugars, LNA monomers, monomers containing arabinose sugars ("ANA monomers”), monomers containing 2'-fluoro- ANA sugars, monomers containing d-arabino-hexitol sugars ( ⁇ monomers”), intercalating monomers as defined in Christensen, Nucl. Acids. Res.
  • nucleoside analogues there is only one of the above types of nucleoside analogues present in the oligomer of the disclosure, or region thereof.
  • the nucleoside analogues contain 2'-0-methoxyethyl-ribose sugars (2'-MOE), or 2'-fluoro-deoxyribose sugars or LNA sugars, and as such the oligonucleotide of the disclosure may comprise nucleoside analogues which are independently selected from these three types of analogue, or may comprise only one type of analogue selected from the three types.
  • 2'-MOE 2'-0-methoxyethyl-ribose sugars
  • LNA sugars 2'-fluoro-deoxyribose sugars or LNA sugars
  • At least one of the nucleoside analogues contains a 2'-MOE-ribose sugar, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleoside analogues containing 2'-MOE-ribose sugars.
  • at least one of the nucleoside analogues contains a 2'-fluoro deoxyribose sugar, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleoside analogues containing 2'-fluoro-deoxyribose sugars.
  • the oligomer according to the disclosure comprises at least one Locked Nucleic Acid (LNA) monomer, such as 1 , 2, 3, 4, 5, 6, 7, or 8 LNA monomers, such as 3-7 or 4-8 LNA monomers, or 3, 4, 5, 6 or 7 LNA monomers.
  • LNA Locked Nucleic Acid
  • all of the nucleoside analogues are LNA monomers.
  • the oligomer comprises both beta-D-oxy-LNA monomers, and one or more of the following LNA units: thio-LNA monomers, amino-LNA monomers, oxy-LNA monomers, and/or ENA monomers in either the beta-D or alpha-L configuration or combinations thereof.
  • the cytosine base moieties of all LNA monomers in the oligomer are 5-methylcytosines.
  • the oligomer comprises both LNA and DNA monomers. Typically, the combined total of LNA and DNA monomers is 10-25, preferably 10-20, even more preferably 12-16.
  • the oligomer, or a region thereof consists of at least one LNA monomer, and the remaining monomers are DNA monomers.
  • the oligomer comprises only LNA monomers and nucleosides (such as RNA or DNA monomers, most preferably DNA monomers), optionally linked with modified linkage groups such as phosphorothioate.
  • nucleosides or nucleoside analogues of the oligomer have one of the naturally occurring bases found in DNA or RNA, that is, adenine, guanine, cytosine, thymidine, or uracil.
  • nucleoside analogues of the oligomer have a base that does not naturally occur in DNA or RNA, or that is a modified base, non-limiting examples of which include xanthine, hypoxanthine, 5-methylcytosine, isocytosine, pseudoisocytosine, 5- bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2- chloro-6-aminopurine.
  • LNA Monomers include xanthine, hypoxanthine, 5-methylcytosine, isocytosine, pseudoisocytosine, 5- bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2- chloro-6-aminopurine.
  • LNA monomer refers to a nucleoside analogue containing a bicyclic sugar (an “LNA sugar”).
  • LNA oligomer and LNA oligonucleotide refer to an oligomer containing one or more LNA monomers.
  • the LNA monomer used in the oligomers of the disclosure has the following structure (formula I):
  • X is selected from— O— ,— S— ,— N(R N )— ,— C(R 6 R 6* )— ;
  • B is selected from hydrogen, optionally substituted C ⁇ -alkoxy, optionally substituted C 1-4 -alkyl, optionally substituted C ⁇ -acyloxy, nucleobases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands;
  • P designates the radical position for an internucleoside linkage to a succeeding monomer, or a 5'-terminal group, such internucleoside linkage or 5'-terminal group optionally including the substituent R 5 or equally applicable the substituent R 5* ;
  • P* designates an internucleoside linkage to a preceding monomer, or a 3'-terminal group
  • each of the substituents R 1 * , R 2 , R 3 , R 5 , R 5* , R 6 and R 6* , which are present is independently selected from hydrogen, optionally substituted C 1 -12 -alkyl, optionally substituted C 2 -i 2 -alkenyl, optionally substituted C 2 . 12 -alkynyl, hydroxy, C ⁇ -alkoxy, C 2 . 12 -alkoxyalkyl, C 2 .
  • asymmetric groups may be found in either R or S orientation.
  • R 1* , R 2 , R 3 , R 5 , R 5* are independently selected from the group consisting of hydrogen, halogen, C 1-6 alkyl, substituted C 1 -6 alkyl, C 2 . 6 alkenyl, substituted C 2 . 6 alkenyl, C 2 . 6 alkynyl or substituted C 2 . 6 alkynyl, alkoxyl, substituted C 1 -6 alkoxyl, acyl, substituted acyl, C 1 -6 aminoalkyl or substituted aminoalkyl.
  • R 1 * , R 2 , R 3 , R 5 , R 5* are hydrogen. For all chiral centers, asymmetric groups may be found in either R or S orientation.
  • R 1 * , R 2 , R 3 are independently selected from the group consisting of hydrogen, halogen, alkyl, substituted C 1 -6 alkyl, C 2 . 6 alkenyl, substituted C 2 . 6 alkenyl, C 2 . 6 alkynyl or substituted C 2 . 6 alkynyl, alkoxyl, substituted C 1 -6 alkoxyl, acyl, substituted acyl, C 1 -6 aminoalkyl or substituted C 1 -6 aminoalkyl.
  • R 1 * , R 2 , R 3 are hydrogen. For all chiral centers, asymmetric groups may be found in either R or S orientation.
  • either R 5 or R 5* are hydrogen, whereas the other group (R 5 or R 5* respectively) is selected from the group consisting of C 1-5 alkyl, C 2 . 6 alkenyl, C 2 . 6 alkynyl, substituted C 1 -6 alkyl, substituted C 2 . 6 alkenyl, substituted C 2 .
  • each substituted group is mono or poly substituted with substituent groups independently selected from halogen, C 1 -6 alkyl, substituted C 1-6 alkyl, C 2 . 6 alkenyl, substituted C 2 . 6 alkenyl, C 2 . 6 alkynyl, substituted C 2 .
  • each ⁇ and J 2 is, independently, H, C 1-6 alkyl, substituted C 1 -6 alkyl, C 2 . 6 alkenyl, substituted C 2 . 6 alkenyl, C 2 . 6 alkynyl, substituted C 2 . 6 alkynyl, C 1 -6 aminoalkyl, substituted aminoalkyl or a protecting group.
  • R 5 or R 5* is a substituted alkyl.
  • each ⁇ and J 2 is, independently H or C 1-6 alkyl.
  • either R 5 or R 5* is methyl, ethyl or methoxymethyl. In some embodiments either R 5 or R 5* is methyl.
  • B is a nucleobase, including nucleobase analogues and naturally occurring nucleobases, such as a purine or pyrimidine, or a substituted purine or substituted pyrimidine.
  • B can be a nucleobase selected from the group consisting of adenine, cytosine, thymine, adenine, uracil, or a modified or substituted nucleobase, such as 5-thiazolo-uracil, 2-thio-uracil, 5-propynyl-uracil, 2-thio- thymine, 5-methylcytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, and 2,6-diaminopurine.
  • R 4* and R 2* together designate a biradical selected from -C(R A R B )-0-, -C(R A R B )-C(R C R D )-0-, -C(R A R B )-C(R C R D )-C(R E R F )-0-, -C(R A R B )-0-C(R C R D )-, - - CCR'R NCR 0 )-, -C(R A R B )-C(R C R D )- N(R E )-, -C(R A R B )-N(R C )-0-, and -C(R A R B )-S-, -C(R A R B )- C(R C R D )-S-, wherein R A , R B , R C , R D , R E , and R F each is independently selected from hydrogen, substituted C 1 -12
  • R 4* and R 2* together designate the biradical C(R a R b )-N(R c )-0-, wherein R a and R b are independently selected from the group consisting of hydrogen, halogen, C 1 -6 alkyl, substituted alkyl, C 2 _ 6 alkenyl, substituted C 2 _ 6 alkenyl, C 2 _ 6 alkynyl or substituted C 2 .
  • R c is selected from the group consisting of hydrogen, halogen, C 1-6 alkyl, substituted C 1 -6 alkyl, C 2 . 6 alkenyl, substituted C 2 . 6 alkenyl, C 2 . 6 alkynyl or substituted C 2 . 6 alkynyl, alkoxyl, substituted C 1 -6 alkoxyl, acyl, substituted acyl, C 1 -6 aminoalkyl or substituted aminoalkyl, such as hydrogen.
  • R c is selected from the group consisting of hydrogen, halogen, C 1-6 alkyl, substituted C 1 -6 alkyl, C 2 . 6 alkenyl, substituted C 2 . 6 alkenyl, C 2 . 6 alkynyl or substituted C 2 . 6 alkynyl, alkoxyl, substituted C 1 -6 alkoxyl, acyl, substituted acyl, C 1 -6 aminoalkyl or substituted aminoalkyl, such as hydrogen.
  • R 4* and R 2* together designate the biradical C(R a R b )-0-C(R c R d ) -0-, wherein R a , R b , R c , and R d are independently selected from the group consisting of hydrogen, halogen, alkyl, substituted C 1 -6 alkyl, C 2 . 6 alkenyl, substituted C 2 . 6 alkenyl, C 2 . 6 alkynyl or substituted C 2 .
  • R 4* and R 2* form the biradical -CH(Z)-0-, wherein Z is selected from the group consisting of alkyl, C 2 . 6 alkenyl, C 2 . 6 alkynyl, substituted C 1 -6 alkyl, substituted C 2 . 6 alkenyl, substituted C 2 .
  • Z is C ⁇ e alkyl or substituted C 1 -6 alkyl.
  • Z is methyl. In some embodiments Z is substituted C 1-6 alkyl. In some embodiments said substituent group is C 1 -6 alkoxy. In some embodiments Z is CH 3 OCH 2 -.
  • asymmetric groups may be found in either R or S orientation. Exemplary bicyclic nucleotides are disclosed in US 7,399,845.
  • R 1 * , R 2 , R 3 , R 5 , R 5* are hydrogen.
  • R 1* , R 2 , R 3 * are hydrogen, and one or both of R 5 , R 5* may be other than hydrogen as referred to above and as disclosed in WO 2007/134181 . For all chiral centers, asymmetric groups may be found in either R or S orientation.
  • R 4* and R 2* together designate a biradical which comprise a substituted amino group in the bridge such as consist or comprise of the biradical - CH 2 -N(R C )-, wherein R c is Ci _ i 2 alkyloxy.
  • R 1* , R 2 , R 3 , R 5 , R 5* are independently selected from the group consisting of hydrogen, halogen, C 1-6 alkyl, substituted C 1 -6 alkyl, C 2 . 6 alkenyl, substituted C 2 . 6 alkenyl, C 2 . 6 alkynyl or substituted C 2 . 6 alkynyl, C ⁇ alkoxyl, substituted C 1 -6 alkoxyl, acyl, substituted acyl, C 1 j3 aminoalkyl or substituted C ⁇ aminoalkyl.
  • R 1 * , R 2 , R 3 , R 5 , R 5* are hydrogen.
  • R 1 * , R 2 , R 3 are hydrogen and one or both of R 5 , R 5* may be other than hydrogen as referred to above and in WO 2007/134181 .
  • R 1 * , R 2 , R 3 , R 5 , R 5* are hydrogen.
  • Exemplary bicyclic nucleotides are disclosed in WO2008/154401 .
  • R 1 * , R 2 , R 3 , R 5 , R 5* are independently selected from the group consisting of hydrogen, halogen, C 1 -6 alkyl, substituted C 1 -6 alkyl, C 2 _ 6 alkenyl, substituted C 2 _ 6 alkenyl, C 2 . 6 alkynyl, substituted C 2 . 6 alkynyl, C ⁇ alkoxyl, substituted C ⁇ alkoxyl, acyl, substituted acyl, C 1 -6 aminoalkyl or substituted C 1-6 aminoalkyl.
  • R 1 * , R 2 , R 3 , R 5 , R 5* are hydrogen.
  • R 1* , R 2 , R 3 are hydrogen and one or both of R 5 , R 5* may be other than hydrogen as referred to above and in WO 2007/134181 or WO2009/067647 (disclosing certain alpha-L-bicyclic nucleic acids analogs), each of which is incorporated by reference in its entirety. For all chiral centers, asymmetric groups may be found in either R or S orientation.
  • LNA monomers have a structure selected from the followin group (from the left, formula II, formula III, and formula IV, respectively):
  • B is a nucleobase, including nucleobase analogues and naturally occurring nucleobases, such as a purine or pyrimidine, or a substituted purine or substituted pyrimidine.
  • B can be a nucleobase selected from the group consisting of adenine, cytosine, thymine, adenine, uracil, or a modified or substituted nucleobase, such as 5-thiazolo-uracil, 2-thio-uracil, 5-propynyl-uracil, 2-thio-thymine, 5-methylcytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, and 2,6- diaminopurine.
  • asymmetric groups may be found in either R or S orientation.
  • LNA monomers have the following structure (formula V):
  • Y is selected from the group consisting of -0-, -CH 2 0-, -S-, -NH-, N(R e ) and -CH 2 -;
  • Z and Z* are independently selected from the group consisting of an internucleotide linkage, R H , a terminal group and a protecting group, wherein R H is selected from hydrogen and C 1 _ -alkyl; B is a natural or non-natural nucleobase; and
  • R a , R b R c , R d and R e are independently selected from the group consisting of hydrogen, substituted C 1 -12 -alkyl, substituted C 2 -i 2 -alkenyl, substituted C 2 . 12 -alkynyl, hydroxy, C ⁇ -alkoxy, C 2 . 12 -alkoxyalkyl, C 2 .
  • asymmetric groups may be found in either R or S orientation.
  • Two exemplary stereochemical isomers of formula V include the following beta-D and alpha-L configurations (from the left, formula VI and formula VII, respectively),
  • thio-LNA refers to an LNA monomer in which Y in formula VI or formula VII above is either— S— or— CH 2 — S— .
  • a thio-LNA monomer can be in either the beta-D or alpha-L-configuration.
  • amino-LNA refers to an LNA monomer in which Y in formula VI or formula VII above is selected from— N(H)— , N(R)— , CH 2 — N(H)— , and— CH 2 — N(R)— , where R is selected from hydrogen and C 1 _ -alkyl.
  • An amino-LNA monomer can be in either the beta-D or the alpha-L-configuration.
  • C ⁇ -alkyl means a linear or branched saturated hydrocarbon chain wherein the chain has from one to four carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.
  • oxy-LNA refers to an LNA monomer in which Y in formula VI or formula VII above is either— O— or— CH 2 — O— .
  • An oxy-LNA monomer can be in either the beta-D or the alpha-L-configuration.
  • ⁇ refers to an LNA monomer in which Y in formula VI or formula VII above is— CH 2 — O— (where the oxygen atom of— CH 2 — O— is attached to the 2'-position relative to the base B).
  • the LNA monomer is selected from a beta-D-oxy-LNA monomer, an alpha-L-oxy-LNA monomer, a beta-D-amino-LNA monomer and a beta-D-thio- LNA monomer, in particular a beta-D-oxy-LNA monomer.
  • oligomers of the disclosure are capable of recruiting an endoribonuclease, such as Ribonuclease H (RNase H), to RNA targets to which the oligomers hybridize.
  • RNase H is an endoribonuclease that specifically hydrolyzes the phosphodiester bonds of RNA that is hybridized to DNA.
  • RNase H operates to hydrolyze and eliminate the RNA target, such as a targeted mRNA.
  • RNase H Ribonuclease H
  • Other mechanisms of action are possible.
  • certain oligomers of the disclosure may also be capable of preventing translation of target mRNAs to which the oligomers have hybridized through steric hindrance of the translation machinery.
  • an oligomer comprises a region of at least 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 contiguous monomers which, after forming a duplex with a target region of a target RNA, contributes to recruiting RNase, such as RNase H.
  • the oligomer region capable of recruiting RNAse may be region B, as that term is defined elsewhere herein.
  • the region of the oligomer capable of recruiting RNAse H e.g., region B, consists of 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 monomers.
  • US 2004-0102618 A1 provides exemplary, non-limiting, in vitro methods for measuring RNaseH activity that may be used to determine if oligomers of the disclosure can recruit RNaseH.
  • an oligomer is deemed capable of recruiting RNase H if the initial rate of RNase H activity (pmol/L/min) against a duplex containing an RNA target and complementary test oligomer is at least about 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the RNase H activity against the same RNA target bound by an oligonucleotide containing the same base sequence, but in which all monomers are DNA, there are no 2' substitutions, and all internucleoside linkages are phosphorothioate.
  • the region of the oligomer responsible for recruiting RNase H after forming a duplex with a target RNA comprises or consists of DNA monomers only, or comprises in addition LNA monomers.
  • the LNA monomers are in the alpha-L configuration, a non-limiting example of which is alpha-L-oxy LNA.
  • An oligomer of the disclosure may comprise nucleosides and nucleoside analogues, each alone or in combination, and may be a gapmer, a headmer, a mixmer (as those terms are defined herein), or some other configuration.
  • a "headmer” is defined as an oligomer that comprises a first region and a second region of contiguously linked monomers, where the 5'-most monomer of the second region is linked to the 3'-most monomer of the first region, the first region comprising a contiguous stretch of linked nucleoside analogues relatively incapable of recruiting RNase H and the second region comprising a contiguous stretch of linked monomers, for example, DNA monomers, capable of recruiting RNase H after hybridizing to a target RNA.
  • a "tailmer” is defined as an oligomer that comprises a first region and a second region of contiguously linked monomers, where the 5'-most monomer of the second region is linked to the 3'-most monomer of the first region, the first region comprising a contiguous stretch of linked monomers, for example, DNA monomers, capable of recruiting RNase H after hybridizing to a target RNA and the second region comprising a contiguous stretch of linked nucleoside analogues relatively incapable of recruiting RNase H.
  • a "mixmer” is an oligomer comprising contiguously linked monomers of both types, i.e., monomers that are capable of recruiting RNase H (if not alone, then contiguously linked with similar monomers), e.g., DNA monomers, and monomers relatively incapable of recruiting RNase H, where monomers of each type are not segregated into separate regions.
  • mixmers comprise monomers of both types in an alternating pattern that may be regular or irregular.
  • the oligomer of the disclosure is a gapmer.
  • a "gapmer” is an oligomer comprising at least one stretch of contiguously linked monomers capable of recruiting an RNAse (for example, RNAseH) when the oligomer forms a duplex with a complementary RNA molecule (such as a mRNA target).
  • monomers capable of recruiting RNAse include DNA monomers, alpha-L-LNA monomers, C4' alkylayted DNA monomers (as described further in WO/2009/090182 and Vester et al., Bioorg. Med. Chem. Lett. 18 (2008) 2296 - 2300,), and unlocked nucleic acid (UNA) in which the bond between the ribose C2' and C3' atoms is cleaved as described in Fluiter et al., Mol. Biosyst., 5:838-843 (2009).
  • DNA monomers alpha-L-LNA monomers
  • C4' alkylayted DNA monomers as described further in WO/2009/090182 and Vester et al., Bioorg. Med. Chem. Lett. 18 (2008) 2296 - 2300,
  • UPA unlocked nucleic acid
  • a gapmer consists of three regions, A-B-C, contiguously arranged from 5' to 3'.
  • Region A may also be called a 5' wing or flanking segment or region
  • region B may also be called a gap or central segment or region
  • region C may also be called a 3' wing or flanking segment or region.
  • region A consists of 1 , 2, 3, 4, 5 or 6 contiguously linked nucleoside analogues, such as an affinity-enhancing nucleoside analogue.
  • region C consists of 1 , 2, 3, 4, 5 or 6 contiguously linked nucleoside analogues, such as an affinity-enhancing nucleoside analogue.
  • regions A and C each consist of 1 , 2, 3, 4, 5 or 6 contiguously linked nucleoside analogues.
  • any one or more of the nucleoside analogs of region A and/or region C can be a LNA monomer. In some embodiments, all the monomers of region A and/or region C are LNA monomers.
  • region B consists of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 contiguously linked monomers capable of recruiting an RNAse, for example RNAse H, when the gapmer forms a duplex with a target RNA, for example mRNA.
  • any one or more of the monomers of region B is a DNA monomer.
  • any one or more of the monomers of region B is a nucleotide.
  • any one or more of the monomers of region B is a DNA nucleotide.
  • all monomers of region B are DNA nucleotides.
  • a gapmer optionally includes an additional region D positioned 5' to region A (that is, D-A-B-C), or positioned 3' of region C (that is, A-B-C-D).
  • regions A and/or C can each independently consist of 1 , 2, 3, 4, 5 or 6 contiguously linked nucleoside analogues, any one or more of which can be a LNA monomer
  • region B consists of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 contiguously linked monomers capable of recruiting an RNAse, any one or more of which can be a DNA monomer, such as a DNA nucleotide
  • region D consists of 1 , 2 or 3 contiguously linked monomers, for example, DNA monomers.
  • Exemplary gapmers of the disclosure include those having the following structure, where the first position corresponds to the number of contiguously linked nucleoside analogues in region A, the second position corresponds to the number of contiguously linked DNA monomers in region B, and the third position corresponds to the number of contiguously linked nucleoside analogues in region C: 1 -8-1 , 1 -8-2, 2-8-1 , 2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-1 , 4-8-2, 1 -8-4, 2-8-4, 1 -9-1 , 1 -9-2, 2-9-1 , 2-9-2, 2-9-3, 3-9-2, 1-9-3, 3-9-1 , 4-9-1 , 1 -9-4, or; 1 -10-1 , 1 -10-2, 2-10-1 , 2-10-2, 1 -10-3, 3-10-1 , 3-10-3, 3-10-4, 4-10-3, 3-9-3, 3-9-4, 4-9-3, 3-8-3, 3-8-4, 4, 4-10
  • any one or more of the monomers of region A can be a LNA monomer
  • any one or more of the monomers of region C can be a LNA monomer
  • any one or more of the monomers of region B can be a DNA monomer.
  • each monomer of region A and region C are LNA monomers and each monomer of region B is a DNA monomer.
  • region A and/or region C comprise monomers having a 2'-0-methoxyethyl-ribose sugar (2' MOE) or monomers having a 2'-fluoro-deoxyribose sugar.
  • region B comprises at least one LNA monomer in the alpha-L configuration. In certain embodiments, region B comprises at least one alpha-L-oxy LNA monomer. In other embodiments, all the LNA monomers in region B are alpha-L-oxy LNA monomers.
  • a gapmer can be a "shortmer,” defined as a gapmer 10, 1 1 , 12, 13 or 14 contiguously linked monomers in length.
  • the monomers of the oligomers, including gapmers, described herein are coupled together into a contiguously linked sequence via linkage groups. Each monomer (except for the monomer occupying the 3'-most position in an oligomer) is linked to a 3'- adjacent monomer via a linkage group.
  • linkage group or "internucleoside linkage” mean a chemical group capable of covalently coupling together two contiguous monomers. Specific non-limiting examples include phosphate groups (forming a phosphodiester between adjacent nucleoside monomers) and phosphorothioate groups (forming a phosphorothioate linkage between adjacent nucleoside monomers).
  • nuclease resistant internucleoside linkages can be used in place of more nuclease sensitive linkages.
  • a phosphorothioate or boranophosphate linkage can be used in place of a phosphodiester linkage group.
  • the former linkage chemistries are more resistant to endo and exonucleases but are still capable of activating RNAse H, thereby permitting RNAse H-mediated antisense inhibition of expression of a target gene.
  • linkage groups containing sulphur can be used.
  • S sulphur
  • phosphorothioate linkage groups can be used to link together adjacent monomers of the oligomer, particularly for the gap region (that is, region B) of gapmers.
  • phosphorothioate linkages can be used to link together monomers in the flanking A and C regions as well.
  • phosphorothioate linkages can be used for linking regions A or C to a region D, as well as for linking together monomers within region D if present.
  • regions A, B and C comprise linkage groups other than phosphorothioate, such as phosphodiester linkages, particularly, for instance when the use of certain nucleoside analogues acts to protect sensitive linkage groups within regions A and C from endo or exonuclease digestion, for example, when regions A and C comprise LNA monomers.
  • phosphodiester linkages for example one, two or three linkages, into an oligomer with a predominantly phosphorothioate backbone, particularly where phosphorothioate groups link nucleoside analogue monomers (for example, in regions A and/or C), can modify the bioavailability and/or bio-distribution of the resulting oligomer. This is described in additional detail in WO2008/1 13832.
  • internucleoside linkage groups are phosphorothioate, whereas in other embodiments the internucleoside linkages of oligomers include both phosphorothioate and phosphodiester linkages.
  • oligomers of the disclosure can comprise at least one bicyclic nucleoside attached to the 3' and/or 5' terminus by a neutral internucleoside linkage.
  • neutrally linked terminal bicyclic nucleosides is described in additional detail in WO 2009/124238.
  • Examples of neutral internucleoside linkages include phosphotriester, a methylphosphonate, methylene(methylimino) or MMI, amide-3, formacetal or thioformacetal.
  • the remaining linkages in the oligomer may be phosphorothioate or some other type of internucleoside linkage. Conjugates
  • conjugate is intended to indicate a heterogenous molecule formed by the covalent attachment (“conjugation") of the oligomer as described herein to one or more non-nucleotide, or non-polynucleotide moieties.
  • non-nucleotide or non- polynucleotide moieties include macromolecular agents such as proteins, fatty acid chains, sugar residues, glycoproteins, polymers, or combinations thereof.
  • proteins may be antibodies for a target protein.
  • Typical polymers may be polyethylene glycol.
  • the oligomer of the invention may comprise both a polynucleotide region which typically consists of a contiguous sequence of nucleotides, and a further non-nucleotide region.
  • the compound may comprise non-nucleotide components, such as a conjugate component.
  • the oligomeric compound is linked to ligands/conjugates, which may be used, e.g. to increase the cellular uptake of oligomeric compounds.
  • ligands/conjugates which may be used, e.g. to increase the cellular uptake of oligomeric compounds.
  • WO2007/031091 provides suitable ligands and conjugates, and is hereby incorporated by reference in its entirety.
  • the invention also provides for a conjugate comprising the compound according to the invention as herein described, and at least one non-nucleotide or non-polynucleotide moiety covalently attached to said compound. Therefore, in various embodiments where the compound of the invention consists of a specified nucleic acid or nucleotide sequence, as herein disclosed, the compound may also comprise at least one non-nucleotide or non- polynucleotide moiety (e.g. not comprising one or more nucleotides or nucleotide analogues) covalently attached to said compound.
  • Conjugation may be performed in order to enhance or otherwise alter the activity, cellular distribution or cellular uptake of the oligomer of the invention.
  • moieties include, but are not limited to, antibodies, polypeptides, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g.
  • a phospholipids e.g., di-hexadecyl- rac-glycerol or triethylammonium 1 ,2-di-o-hexadecyl-rac-glycero-3-h-phosphon
  • the oligomers of the invention may also be conjugated to active drug substances, for example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • active drug substances for example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • the conjugated moiety is a sterol, such as cholesterol.
  • the conjugated moiety comprises or consists of a positively charged polymer, such as a positively charged peptides of, for example from 1 -50, such as 2 - 20 such as 3 - 10 amino acid residues in length, and/or polyalkylene oxide such as polyethylglycol(PEG) or polypropylene glycol (see, for example, WO 2008/034123, which is hereby incorporated by reference in its entirety).
  • a positively charged polymer such as a polyalkylene oxide may be attached to the oligomer of the invention via a linker such as the releasable inker described in WO 2008/034123.
  • conjugate moieties may be used in the conjugates of the invention: ER -3'
  • activated oligomer refers to an oligomer of the disclosure that is covalently linked to at least one functional moiety that permits covalent linkage of the oligomer to one or more moieties to form a conjugate as described above.
  • Functional moieties are chemical groups that can be covalently bonded to the oligomer.
  • a functional moiety can be bonded to an oligomer via a 5' or 3' terminal group, or chemical group located elsewhere in an oligomer.
  • Non-limiting examples include amino, sulfhydryl or hydroxyl groups, for example, a 3'-hydroxyl group or an exocyclic NH 2 group of an adenine base.
  • a functional group can be bonded to a spacer that in turn is bonded to the oligomer.
  • terminal and other groups are unprotected, whereas in some other embodiments, the terminal or other group is protected, for example, by any suitable protecting group such as those described in "Protective Groups in Organic Synthesis” by Theodora W Greene and Peter G M Wuts, 3rd edition (John Wiley & Sons, 1999).
  • Non-limiting examples of hydroxyl protecting groups include esters such as acetate ester, or aralkyl groups such as benzyl, diphenylmethyl, triphenylmethyl, or tetrahydropyranyl groups.
  • Non-limiting examples of amino protecting groups include benzyl, alpha-methylbenzyl, diphenylmethyl, triphenylmethyl, benzyloxycarbonyl, or tert-butoxycarbonyl groups, and acyl groups such as trichloroacetyl or trifluoroacetyl groups.
  • the functional moiety is self-cleaving. In other embodiments, the functional moiety is biodegradable. See e.g., U.S. Pat. No. 7,087,229, which is incorporated herein by reference in its entirety.
  • oligomers of the disclosure are functionalized at the 5' end in order to allow covalent attachment of a moiety to the 5' end of the oligomer.
  • oligomers of the disclosure can be functionalized at the 3' end to allow covalent attachment of a moiety to the 3' end of the oligomer.
  • oligomers of the disclosure can be functionalized along the backbone or on the heterocyclic base moiety.
  • oligomers of the disclosure can be functionalized at more than one position independently selected from the 5' end, the 3' end, the backbone and the base.
  • activated oligomers of the disclosure are synthesized by incorporating during the synthesis one or more monomers that are covalently attached to a functional moiety. In other embodiments, activated oligomers of the disclosure are synthesized with unfunctionalized monomers and the oligomer is functionalized after completion of synthesis.
  • the oligomers are functionalized with a hindered ester containing an aminoalkyl linker, wherein the alkyl portion has the formula (CH 2 ) W , wherein w is an integer ranging from 1 to 10, for example 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group is attached to the oligomer via an ester group (-0-C(0)-(CH 2 ) w NH).
  • the oligomers are functionalized with a hindered ester containing a (CH 2 ) w -sulfhydryl (SH) linker, wherein w is an integer ranging from 1 to 10, for example 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group attached to the oligomer via an ester group (-O-C(O)- (CH 2 ) W SH).
  • a hindered ester containing a (CH 2 ) w -sulfhydryl (SH) linker wherein w is an integer ranging from 1 to 10, for example 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group attached to the oligomer via an ester group (-O-C(O)- (CH 2 ) W SH).
  • sulfhydryl-activated oligonucleotides are conjugated with polymer moieties such as polyethylene glycol or peptides via formation of a disulfide bond.
  • Activated oligomers covalently linked to at least one functional moiety can be synthesized by any method known in the art, including those disclosed in U.S. Patent Publication No. 2004/0235773 and in Zhao et al. (2007) J. Controlled Release 1 19:143-152; and Zhao et al. (2005) Bioconjugate Chem. 16:758-766.
  • oligomers of the disclosure can be functionalized by introducing sulfhydryl, amino or hydroxyl groups into the oligomer using substantially linear functionalizing reagents having a phosphoramidite at one end linked through a hydrophilic spacer chain to the opposing end which comprises a protected or unprotected sulfhydryl, amino or hydroxyl group.
  • substantially linear functionalizing reagents having a phosphoramidite at one end linked through a hydrophilic spacer chain to the opposing end which comprises a protected or unprotected sulfhydryl, amino or hydroxyl group.
  • such activated oligomers have a functionalizing reagent coupled to a 5'-hydroxyl group of the oligomer. In other embodiments, the activated oligomers have a functionalizing reagent coupled to a 3'-hydroxyl group. In still other embodiments, the activated oligomers of the disclosure have a functionalizing reagent coupled to a hydroxyl group on the backbone of the oligomer.
  • the 5'-terminus of an oligomer bound to a solid-phase is functionalized with a dienyl phosphoramidite derivative followed by conjugation of the deprotected oligomer with, e.g., an amino acid or peptide via a Diels-Alder cycloaddition reaction.
  • incorporation of monomers containing 2'-sugar modifications, such as a 2'-carbamate substituted sugar or a 2'-(0-pentyl-N-phthalimido)- deoxyribose sugar into the oligomer facilitates covalent attachment of conjugated moieties to the sugars of the oligomer.
  • an oligomer with an amino-containing linker at the 2'-position of one or more monomers is prepared using a reagent such as, for example, 5'-dimethoxytrityl-2'-0-(e-phthalimidylaminopentyl)-2'-deoxyadenosine-3'-N,N-diisopropyl- cyanoethoxy phosphoramidite. See, e.g., Manoharan, et al., Tetrahedron Letters, 32:7171 (1991).
  • oligomers of the disclosure can be provided with amine-containing functional moieties on the nucleobase, including on the N6 purine amino groups, on the exocyclic N2 of guanine, or on the N4 or 5 positions of cytosine.
  • such functionalization may be achieved during oligomer synthesis by using a commercial reagent that is already functionalized.
  • Certain functional moieties are available from commercial sources.
  • heterobifunctional and homobifunctional linking moieties are available from the Pierce Co. (Rockford, III.)
  • 5'-Amino-Modifier C6 reagents are available from Glen Research Corporation (Sterling, Va.) and from ABI (Applied Biosystems Inc., Foster City, Calif.) as Aminolink-2
  • 3'-Amino-Modifier reagents are available from Glen Research Corporation (Sterling, Va.) and from Clontech Laboratories Inc. (Palo Alto, Calif.).
  • compositions comprising Oligomers
  • compositions comprising oligomers or conjugates.
  • Oligomers of the disclosure may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations.
  • Pharmaceutical compositions of the disclosure include, but are not limited to, solutions, suspensions, emulsions, and liposome-containing formulations.
  • Suspensions can be prepared using aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, carboxymethylcellulose, sorbitol or dextran.
  • compositions of the disclosure can also include a pharmaceutically acceptable carrier diluent or solvent, such as sterile water, alcohol, saline or phosphate-buffered saline (PBS).
  • a pharmaceutically acceptable carrier diluent or solvent such as sterile water, alcohol, saline or phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • Such diluents are useful, for example, in compositions to be delivered parenterally, for example, intrathecally or intravenously.
  • compositions of the disclosure can also include one or more pharmaceutically acceptable excipients.
  • Excipients are selected so as to provide for certain desired properties of the composition, such as stability, bioavailability, or pharmacokinetics of the active agent, and ease of administration according to the desired mode. See, e.g., Martin, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042).
  • Compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form.
  • Exemplary pharmaceutical excipients include, but are not limited to, carrier substances, such as polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4' isothiocyano-stilbene-2,2'-disulfonic acid, fillers, bulking substances, such as an inert bulk nucleic acid, or stabilizers, such as lactose, sucrose, mannitol and other sugars, pH buffers, such as Tris-HCI, acetate, or phosphate, detergents, surfactants, including ionic and non-ionic surfactants, such as polysorbate 80, solubilizing agents, wetting agents, anti-oxidants, such as ascorbic acid or sodium metabisulfite, preservatives, such as thimersol or benzyl alcohol, and chelating agents.
  • carrier substances such as polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido
  • compositions can include agents that enhance uptake of oligonucleotides at the cellular level.
  • agents that enhance uptake of oligonucleotides at the cellular level For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Dello et al., PCT Application WO 1997/30731), have been demonstrated to enhance the cellular uptake of oligonucleotides.
  • agents that can be added to enhance the penetration of oligomers include glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
  • oligomers of the disclosure can be used in unmodified form, or can be modified to create prodrugs, pharmaceutically acceptable acid and base addition salts, esters, or salts of such esters, of such oligomers. See, for example, Berge, et al., "Pharmaceutical Salts," J. of Pharm. Sci., 1977, 66, 1 -19.
  • a prodrug can include the incorporation of additional nucleosides or nucleotides at one or both ends of an oligomer that are cleaveable by endogenous nucleases within a subject's body to form the biologically active oligomer.
  • Prodrug versions of oligomers of the disclosure can be prepared as SATE [(S-acetyl-2-thioethyl)phosphate] derivatives according to the methods disclosed in WO 1993/24510, WO 1994/26764 and U.S. Pat. No. 5,770,713.
  • the term “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the oligomer while at the same time exhibiting acceptable levels of undesired toxic effects.
  • Pharmaceutically acceptable base addition salts of the oligomers of the disclosure can be formed with basic compounds, such as metal cations, such as alkali and alkaline earth metals, ammonium and quaternary ammonium cations, or amines, such as organic amines.
  • metal cations include sodium, potassium, magnesium, calcium, zinc, bismuth, barium, aluminum, copper, cobalt, nickel, or cadmium.
  • Non-limiting examples of organic amines are ⁇ , ⁇ '-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, D- glucosamine, tetraethylammonium, and procaine.
  • Polyamines such as spermine and spermidine can also be used.
  • Base addition salts can be prepared by contacting the free acid form of a compound, for example, an oligomer, with a sufficient amount of the desired base to produce the salt. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid.
  • Pharmaceutically acceptable acid addition salts of the oligomers of the disclosure can be formed with organic or inorganic acids.
  • exemplary inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, or nitric acid.
  • exemplary organic acids include carboxylic, sulfonic, sulfo or phospho acids, or N-substituted sulfamic acids.
  • More specific examples include acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, methanesulfonic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid, isonicotinic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1 ,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenes
  • compositions comprising oligomers of the disclosure can be administered together with at least a second agent to treat diseases caused by expansion of the C90RF72 hexameric repeat region.
  • the second agent is a second oligomer of the disclosure which can be included in the same or a different composition as the first oligomer of the disclosure.
  • compositions may be prepared in unit dosage form for the convenience of care providers and to help ensure sterility and reduce wastage.
  • dosage forms can be included in kits that additionally provide a device for administering the composition, such as a syringe and needle for intravenous or intrathecal delivery.
  • the pharmaceutical composition comprising an oligomer of the disclosure is provided in liquid form, and in other embodiments is provided in lyophilized form.
  • a kit can additionally provide a suitable diluent, such as sterile water, saline, or other diluent.
  • Optimal dosing of the oligomers of the disclosure will depend on a variety of factors, including the indication to be treated, the specific biological activity of the oligomer, therapeutic index of the oligomer and the mode of administration. Typically, the chosen dose seeks to maximize efficacy in a particular subject under treatment while minimizing any expected side effects.
  • dosage can be expressed as a certain mass of the oligomer to be administered per unit of mass of the subject being treated.
  • Exemplary dosages include 10 microgram (meg) per kg, 25 mcg/kg, 50 mcg/kg, 75 mcg/kg, 100 mcg/kg, 200 mcg/kg, 300 mcg/kg, 400 mcg/kg, 600 mcg/kg, 700 mcg/kg, 800 mcg/kg, 900 mcg/kg, 250 mcg/kg, 500 mcg/kg, 750 mcg/kg, 1 milligram (mg) per kg, 1 .1 mg/kg, 1 .2 mg/kg, 1 .3 mg/kg, 1 .4 mg/kg, 1 .5 mg/kg, 1 .6 mg/kg, 1 .7 mg/kg, 1 .8 mg/kg, 1 .9 mg/kg, 2 mg/kg, 2.5
  • the mass of oligomer to be administered is not related to mass of the subject, but to some other variable, such as the subject's brain volume or mass, or volume of cerebrospinal fluid.
  • the total mass of oligomer to be administered may be less than if the administration were to be made intravenously or in some other way outside the central nervous system.
  • the disclosure provides for methods of treating or preventing diseases or disorders of the central nervous system associated with expansion of the hexanucleotide repeat region of the C90RF72 gene, or any other gene targeted by an oligomer of the disclosure.
  • the disease is frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS).
  • FTD frontotemporal dementia
  • ALS amyotrophic lateral sclerosis
  • Methods of treatment are carried out by administering a therapeutically effective amount of an oligomer of the disclosure, or composition thereof, to a subject in thereof diagnosed as having the disease or disorder or who is determined to be at greater than normal risk of later developing the disease or disorder.
  • Methods of diagnosis and determinations as to predisposition for developing a disease or disorder are known in the art, including without limitation, observations of behavior, biopsies or genetic tests.
  • Methods are also provided for down-regulating the expression of the C90RF72 gene in a cell or tissue by contacting said cell or tissue with an effective amount of an oligomer of the disclosure, or composition comprising such oligomer.
  • exemplary cells include neurons and exemplary tissues include brain tissue, for example, gray matter (i.e., that portion of brain tissue predominantly comprising the cell bodies of neurons).
  • the disclosure provides for the use of the oligomers of the disclosure for the manufacture of a medicament for the treatment or prevention of a disease or disorder of the central nervous system caused by expansion of the hexanucleotide repeat region of the C90RF72 gene, or any other gene targeted by an oligomer of the disclosure.
  • the disease is frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS).
  • the subject to be treated is a human, and in other embodiments, the subject is a non-human animal, for example, non-human mammal.
  • treatment refers both to treatment of a disease associated with hexanucleotide repeat expansion where symptoms have already presented, as well as prevention of such disease in susceptible subjects who have not experienced a prodrome or more severe symptoms.
  • treatment includes prevention or prophylaxis.
  • the disease to be treated with an oligomer of the disclosure is frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS) associated with hexanucleotide repeat expansion.
  • FTD frontotemporal dementia
  • ALS amyotrophic lateral sclerosis
  • the diseases that may be treated with the instant oligomers are not limited to FTD and ALS, however. Instead, any disease associated with a hexanucleotide repeat expansion that can be targeted with an oligomer of the disclosure can be treated, even if such diseases have not yet been recognized in the art.
  • Treatment involves administering a therapeutically effective amount of an oligomer of the disclosure, or composition thereof.
  • Therapeutic efficacy means any temporary or permanent reduction or amelioration in the severity of symptoms associated with the disease under treatment, or improvement in the condition, functioning or prognosis of the subject with the disease being treated.
  • Therapeutic efficacy can also refer to a reduction in the rate at which a progressive disease worsens, a delay of onset of the first symptoms associated with a disease, or delay before a subject with a lethal disease ultimately succumbs and dies, compared to untreated subjects with the disease.
  • a therapeutically effective amount of an oligomer of the disclosure can reduce muscle weakness associated with ALS, such as in the hands, arms or legs, or in the muscles that control speech, swallowing or breathing.
  • Efficacious treatment can also improve the ability of the subject with ALS to form speech, swallow or breathe, or use his or her arms or legs.
  • Efficacious treatment can also improve mobility, as well as reduce twitching or fasciculation and cramping of the muscles, especially in the hands and feet.
  • Therapeutic efficacy is also associated with a reduction or cessation in the rate at which motor neurons affected by the disease die. Methods for determining amelioration of disease symptoms, including those described herein, may be assessed using methods well-known in the art.
  • a therapeutically effective amount of an oligomer can reduce the extent or rate at which a subject experiences disinhibition, which presents as a loss of restraint in personal relations and social life, as assessed according to methods well-known in the art.
  • FTD initially presents as a movement disorder affecting certain involuntary, automatic muscle functions. These disorders also may impair language and behavior.
  • the two primary FTD movement disorders are corticobasal degeneration (CBD), which causes shakiness, lack of coordination and muscle rigidity and spasms, and progressive supranuclear palsy (PSP), which causes walking and balance problems, frequent falls and muscle stiffness, especially in the neck and upper body, as well as affecting eye movements.
  • CBD corticobasal degeneration
  • PPS progressive supranuclear palsy
  • a therapeutically effective amount of an oligomer can reduce the extent or rate at which the disease process impairs muscle function which may be assessed by methods well-known in the art.
  • a therapeutically effective amount of an oligomer reduces the extent or rate of neurodegeneration caused by FTD or other neurological disorder associated with hexanucleotide repeat expansion.
  • therapeutic efficacy can also be monitored with brain scans, e.g., CAT scan, functional MRI, or PET scan, or other methods well-known in the art.
  • the therapeutic efficacy of an oligomer of the disclosure can also be predicted based on in vitro tests of the ability of the oligomer to reduce the transcription, transcript stability, or translation of a targeted gene. Efficacy can further be assessed by studying the effect of an oligomer on the cells in and behaviors of animal models for a human disease to be treated using methods well-known in the art.
  • a therapeutically effective amount of an oligomer of the disclosure, or composition thereof can be administered as a single dose, or as two or more divided doses separated by predetermined intervals.
  • such intervals include a period of minutes, for example, 5, 10, 15, 30 or more minutes, a period of hours, for example, 1 , 1 .5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20 hours or more, a period of days, for example, 1 , 1 .5, 2, 3, 4, 5, 6 days or more, or a period of weeks, for example, 1 , 2, 3, or more weeks.
  • the invention further provides for an oligomer according to the invention, for use in medicine.
  • the invention further provides for the use of the oligomer of the invention in the manufacture of a medicament for the treatment of one or more of the diseases referred to herein, such as a disease selected from the group consisting of frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS).
  • FDD frontotemporal dementia
  • ALS amyotrophic lateral sclerosis
  • the invention further provides for an oligomer according to the invention, for use for the treatment of one or more of the diseases referred to herein, such as a disease selected from the group consisting of frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS).
  • FDD frontotemporal dementia
  • ALS amyotrophic lateral sclerosis
  • Oligomers of the disclosure can be administered by any mode demonstrated to result in the direct or indirect delivery of a therapeutically effective concentration of oligomers into the brains of subjects to be treated.
  • administration of a composition comprising an oligomer of the disclosure includes parenteral administration, examples of which include administration by the intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intrathecal or intraventricular routes of administration. Administration by any one or more of these routes can be accomplished by injection or infusion through a needle, catheter or cannula, or by some other means.
  • oligomers of the disclosure can be administered intrathecal ⁇ into the cerebrospinal fluid (CSF) by injecting or infusing a composition containing the oligomer under the arachnoid membrane of the brain or spinal cord.
  • CSF cerebrospinal fluid
  • oligomers of the disclosure can be administered into the CSF by intracerebral ventricular (ICV) administration (or simply intraventricular administration) by injecting or infusing a composition containing the oligomer into the brain ventricles through a catheter or cannula.
  • ICV intracerebral ventricular
  • the cannula can be connected to an Ommaya reservoir or osmotic pump implanted under the skin of a subject so that oligomers targeting C90RF72 can be intermittently or continuously infused into the ventricles and CSF over an extended period time, for example, hours, days or weeks.
  • the cannula can be directed into the left or right lateral ventricle, the third ventricle or to the fourth ventricle.
  • ICV has been demonstrated to be an effective way of administering antisense compounds into the brains of test animals. It was demonstrated, for example, that an antisense oligonucleotide targeting superoxide dismutase 1 (SOD1) administered into the ventricles of rats via a cannula reduced both SOD1 mRNA and protein expression throughout the brain and spinal cord. And, in a more recent study, intraventricular administration of an antisense oligonucleotide into mice targeting cellular prion protein was found to delay onset of neurodegenerative disease caused by a pathogenic prion isoform. Smith, et al., J. Clin. Invest.
  • SOD1 superoxide dismutase 1
  • oligomers of the disclosure can be administered directly into the brain ventricles to mix with the cerebrospinal fluid to be transported around the brain and spinal cord and into the parenchyma of these tissues. Oligomers can then be taken up by neurons or other affected cells to target C90RF72 RNA.
  • Reports in the scientific literature suggest that at least on some cases, antisense oligonucleotides administered intravenously are able to cross the blood-brain-barrier and affect gene expression within the central nervous system. See, e.g., Farr et al., J Alzheimers Dis. 2014 Jan 1 ; 40(4):1005-16, and Farr et al., Free Radic Biol Med. 2014 Feb; 67:387-95.
  • Example 1 Treatment with oligomers targeting C90RF72 RNA
  • Cells were cultured in the appropriate medium as described below and maintained at 37°C at 95-98% humidity and 5% C0 2 . Cells were routinely passaged 1 -2 times weekly.
  • ND40063 Primary human skin fibroblast cells isolated from a 41 year old female subject with familial FTD/ALS contains a C90RF72 gene with expanded hexanucleotide repeats. ND40063 cells were purchased from Coriell Institute for Medical Research, and cultured in DMEM (Sigma) with 15% FBS + 100 units/ml penicillin (Gibco) +100 ⁇ g/ml streptomycin (Gibco).
  • HEK-293 Human embryonic kidney cell line purchased from ATCC and cultured in DMEM (Sigma) with 10% FBS + 100 units/ml penicillin (Gibco) +100 ⁇ g/ml streptomycin (Gibco).
  • RNA analysis (5% C0 2 ) in 0.5 ml growth medium supplemented with 15% or 10% FBS, 100 units/ml penicillin (Gibco) and 100 ⁇ g/ml streptomycin (Gibco). Seeding density was 40000 cells/well. Cells were treated in triplicates with different concentrations of oligomers (0.05 ⁇ - 50 ⁇ ) for 3 days. In short, 25 ⁇ (20x) oligomer was added to a total volume of 0.5 ml cell mix per well. Cells were incubated at 37°C for 3 days, then harvested for RNA analysis.
  • First strand synthesis was performed using either random DNA decamers (for total cDNA) or strand-specific primers (sense RT#2 (CAATTCCACCAGTCGCTAGA (SEQ ID NO:177)) or antisense RT#2 (CTGCGGTTGCGGTGCCTGC (SEQ ID NO:178)) together with M-MLV-Reverse Transcriptase (essentially as described by manufacturer (Ambion)).
  • strand-specific primers sense RT#2 (CAATTCCACCAGTCGCTAGA (SEQ ID NO:177)
  • antisense RT#2 CTCGGTTGCGGTGCCTGC (SEQ ID NO:178)
  • M-MLV-Reverse Transcriptase essentially as described by manufacturer (Ambion)
  • the generated cDNA was used in quantitative PCR analysis using a Real-time PCR system from Applied Biosystems. The C9orf72 RNA expression quantified generally as described by the manufacturer. In brief, 4 ⁇ of cDNA was added 6 ⁇ of a mastermix containing Taqman Fast Universal PCR master mix and a primer-probe mix available from Life technologies. All samples were run in duplicates and correlated to a 2-fold dilution series generated from cDNA made from the respective cell line.
  • Relative quantities of C9orf72 RNA were determined from the calculated threshold cycle using the sequence detection software from Applied Biosystems and normalized to the relative quantities of either GAPDH mRNA (qpcr-assay#1) or an internal Mock sample (qpcr-assay#2).
  • Qpcr-assay#1 forward primer: ATCATTTGGGGTTTTGATGG (SEQ ID NO:179):
  • Qpcr-assay#1 detection probe: GTTGGAATGCAGTGATGTCG (SEQ ID NO:181 ) Qpcr-assay#2, forward primer: AAGAGGCGCGGGTAGAAG (SEQ ID NO: 182)
  • Qpcr-assay#2 detection probe: CCCTCTCATTTCTCTGACCG (SEQ ID NO:184)
  • oligomers were designed to target different regions of human C9orf72 RNA. See Table 1 : Oligomers are evaluated for their potential to knockdown C9orf72 pre-mRNA in human ND40063 cells following unassisted uptake. The results showed very potent down regulation of total C9orf72 pre-mRNA (75-80%: ASO-13, ASO-146 and ASO-156) with 15.8 ⁇ of all oligomers.
  • oligomers were tested for their ability to simultaneously target both sense and antisense C9orf72 pre-mRNA strands. See Table 2: Oligomers are evaluated for their potential to knockdown sense or antisense C9orf72 pre-mRNA in human ND40063 cells following unassisted uptake. The results showed very potent down regulation of sense C9orf72 pre-mRNA (80-86%: ASO-9, ASO-154, ASO-156 and ASO-158) and antisense C9orf72 pre-mRNA (76-91 %: ASO-9, ASO-154, ASO-156 and ASO- 158) with 15.8 ⁇ of all oligomers.
  • Example 3 Potent knock-down in cells containing the C9orf72-repeat expansion
  • Column 1 (from left) lists the identification number assigned to each specific oligomer; these identification numbers also correspond to the sequence identification number (SEQ ID NO).
  • Column 2 identifies the nucleobase sequence of each oligomer, including the bases in the 5' flanking region, the gap region and the 3' flanking region.
  • the 5' flanking region and 3' flanking region each consist of one or more beta-D-oxy-LNA monomers identified by capital letters (i.e., A, T, G, C), whereas the gap region consists of DNA monomers identified by lower case letters (i.e., a, t, g, c). All internucleoside linkages are phosphorothioate.
  • certain oligomers described in Table 4 contain cytosine bases modified to be 5-methylcytosine. Specifically, all LNA cytosines in 5' flanking regions and 3' flanking regions (i.e., those denoted by capital "C") are 5-methylcytosine. Furthermore, in the DNA gap regions, any cytosine that immediately precedes a guanine (i.e., those denoted by "eg” or "cG”), are 5-methylcytosine. Other cytosines in gap regions are unmodified. Column 3 reports the maximum C90RF72 RNA reduction effected using 25 ⁇ of each oligomer tested in the primary screen. Where tested, Columns 4, 5, and 6 report IC 50 values for the oligomers.
  • Column 4 reports the concentration in ⁇ of tested oligomers sufficient to reduce C90RF72 total pre-mRNA by 50% (qpcr-assay#1); Column 5 reports the concentration in ⁇ of tested oligomers sufficient to reduce C90RF72 sense pre-mRNA by 50% (qpcr-assay#2); and Column 6 reports the concentration in ⁇ of tested oligomers sufficient to reduce C90RF72 antisense pre-mRNA (qpcr-assay#2).
  • Column 7 indicates the location on the reference genomic DNA (SEQ ID NO:187) to which the oligomer is complementary.
  • Column 8 indicates the location on the reference mRNA (SEQ ID NO:188) to which the oligomer is complementary, if applicable.
  • Columns 9 and 10 provide the number of 5-methylcytosines within the oligomer: Column 9 provides the total number of 5-methylcytosines in the oligomer, and Column 10 provides the number of 5-methylcytosines within the gap region. Column 1 1 provides the total number of locked nucleic acids (LNAs) within the oligomer. Columns 1 1 and 12 provide the lengths of the 5' and 3' flanking regions within each gapmer ("wings"), respectively.
  • LNAs locked nucleic acids
  • GGG acg get gac acA C 0.392 5081 N/A 7 1 5 3 2
  • Example 4 Comparative Inhibition of C90RF72 sense and antisense transcripts by MOE- and LNA-based Gapmers
  • Oligomer 109 is an LNA-containing gapmer consisting of the sequence of SEQ ID NO: 109. The efficacy of this oligomer was compared to a number of gapmers comprising 2'-MOE chemistry, as shown in Table 5.
  • Column 1 lists the sequence identification numbers for the 2'-MOE gapmers used in this Example.
  • Column 2 provides the lengths of the oligomers.
  • Column 3 provides the chemistry and the length of the 5' flanking region (wing), gap region and 3' flanking region (wing), respectively.
  • a 3-10-3 gapmer is 16 nucleosides in length, wherein the central gap segment comprises ten 2'-deoxynucleosides and is flanked by wing segments on both the 5' end and on the 3' end comprising three nucleosides each.
  • Column 4 provides the sequence and structure of the gapmer.
  • Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment comprises a 2'-MOE group.
  • the LNA-based oligomer No. 109 and the 2'-MOE-based oligos listed in Table 5 were tested for the ability to inhibit expression of C90RF72 sense and antisense transcripts.
  • the methods used for this example are essentially as described above, for example in Example 2. Briefly, ND40063 fibroblasts were treated with the respective oligonucleotides by gymnotic delivery at 1 ⁇ , 5 ⁇ and 25 ⁇ , and incubated for 72 hours prior to RNA isolation and analysis as described above. Sense and antisense transcript levels for each treatment were measured and normalized to RNA from mock transfected cells. As shown in Figure 5, the LNA-based Oligomer No. 109 was equally effective in inhibiting expression of both C90RF72 sense and antisense transcripts as several of the 2'-MOE-based oligomers tested.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente divulgation concerne des oligomères capables de cibler l'ARN exprimé à partir du gène C9ORF72 humain contenant une expansion d'une répétition hexanucléotidique pathogène. Ces oligomères sont utiles pour, entre autres, réduire voire éliminer l'ARN du C9ORF72 et/ou les protéines traduites à partir de celui-ci, et pour traiter ou prévenir les maladies ou les troubles provoqués par, ou associés à ladite expansion d'une répétition hexanucléotidique, comprenant la démence fronto-temporale familiale (DFT) et la sclérose latérale amyotrophique familiale (SLA).
PCT/IB2015/056080 2014-08-15 2015-08-10 Oligomères ciblant l'expansion d'une répétition hexanucléotidique dans le gène c9orf72 humain Ceased WO2016024205A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US14/826,829 US20160108396A1 (en) 2014-08-15 2015-08-14 Oligomers targeting hexanucleotide repeat expansion in human c9orf72 gene

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201462037741P 2014-08-15 2014-08-15
US62/037,741 2014-08-15

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US14/826,829 Continuation US20160108396A1 (en) 2014-08-15 2015-08-14 Oligomers targeting hexanucleotide repeat expansion in human c9orf72 gene

Publications (2)

Publication Number Publication Date
WO2016024205A1 true WO2016024205A1 (fr) 2016-02-18
WO2016024205A8 WO2016024205A8 (fr) 2016-10-20

Family

ID=54251541

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2015/056080 Ceased WO2016024205A1 (fr) 2014-08-15 2015-08-10 Oligomères ciblant l'expansion d'une répétition hexanucléotidique dans le gène c9orf72 humain

Country Status (3)

Country Link
US (1) US20160108396A1 (fr)
CA (1) CA2900208A1 (fr)
WO (1) WO2016024205A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9605263B2 (en) 2015-04-16 2017-03-28 Ionis Pharmaceuticals, Inc. Compositions for modulating C9ORF72 expression
WO2017079291A1 (fr) * 2015-11-02 2017-05-11 Ionis Pharmaceuticals, Inc. Composés et méthodes de modulation de c90rf72
US9963699B2 (en) 2012-10-15 2018-05-08 Ionis Pharmaceuticals, Inc. Methods for modulating C9ORF72 expression
US10221414B2 (en) 2013-10-11 2019-03-05 Ionis Pharmaceuticals, Inc. Compositions for modulating C9ORF72 expression
US10443052B2 (en) 2012-10-15 2019-10-15 Ionis Pharmaceuticals, Inc. Compositions for modulating C9ORF72 expression
US10577604B2 (en) 2012-10-15 2020-03-03 Ionis Pharmaceuticals, Inc. Methods for monitoring C9ORF72 expression
EP3664816A4 (fr) * 2017-08-08 2021-05-19 Wave Life Sciences Ltd. Compositions oligonucléotidiques et procédés associés
WO2021205005A3 (fr) * 2020-04-09 2021-11-18 Association Institut De Myologie Séquences antisens pour le traitement de la sclérose latérale amyotrophique
US20220290136A1 (en) * 2020-09-30 2022-09-15 Crispr Therapeutics Ag Materials and methods for treatment of amyotrophic lateral sclerosis
EP4093409A4 (fr) * 2020-01-23 2024-01-10 University of Southern California Antagonisme en tant que thérapie pour des protéinopathies tdp-43

Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2370451B1 (fr) 2008-12-02 2016-11-16 Wave Life Sciences Japan, Inc. Procédé pour la synthèse d'acides nucléiques modifiés au niveau de l'atome de phosphore
KR101885383B1 (ko) 2009-07-06 2018-08-03 웨이브 라이프 사이언시스 리미티드 신규한 핵산 프로드러그 및 그의 사용 방법
US10428019B2 (en) 2010-09-24 2019-10-01 Wave Life Sciences Ltd. Chiral auxiliaries
EP2734208B1 (fr) 2011-07-19 2017-03-01 Wave Life Sciences Ltd. Procédés pour la synthèse d'acides nucléiques fonctionnalisés
PL2872485T3 (pl) 2012-07-13 2021-05-31 Wave Life Sciences Ltd. Asymetryczna grupa pomocnicza
US11162096B2 (en) 2013-10-14 2021-11-02 Ionis Pharmaceuticals, Inc Methods for modulating expression of C9ORF72 antisense transcript
EP4137572A1 (fr) 2014-01-16 2023-02-22 Wave Life Sciences Ltd. Conception chirale
WO2016112132A1 (fr) 2015-01-06 2016-07-14 Ionis Pharmaceuticals, Inc. Compositions destinées à moduler l'expression du transcrit antisens c9orf72
US10407678B2 (en) * 2015-04-16 2019-09-10 Ionis Pharmaceuticals, Inc. Compositions for modulating expression of C9ORF72 antisense transcript
MX381216B (es) * 2016-09-30 2025-03-12 Regeneron Pharma Animales no humanos que tienen una expansion de repeticiones de hexanucleotidos en un locus c9orf72.
WO2019051025A2 (fr) * 2017-09-06 2019-03-14 The Trustees Of Columbia University In The City Of New York Traitement de cancers agressifs par ciblage de c9orf72
EP3701030A4 (fr) * 2017-10-23 2022-04-20 Prevail Therapeutics, Inc. Thérapies géniques pour maladie neurodégénérative
AU2019310040A1 (en) * 2018-07-23 2021-02-11 Enclear Therapies, Inc. Methods of treating neurological disorders
AU2019403015B2 (en) * 2018-12-20 2024-01-18 Regeneron Pharmaceuticals, Inc. Nuclease-mediated repeat expansion
CN114072501A (zh) * 2019-05-06 2022-02-18 马萨诸塞大学 抗c9orf72寡核苷酸及相关方法
JP2025501682A (ja) * 2021-12-03 2025-01-23 クラリス コーポレーション 改変された骨格化学を有するギャップマーアンチセンスオリゴヌクレオチド

Citations (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4914210A (en) 1987-10-02 1990-04-03 Cetus Corporation Oligonucleotide functionalizing reagents
US4962029A (en) 1987-10-02 1990-10-09 Cetus Corporation Covalent oligonucleotide-horseradish peroxidase conjugate
WO1993024510A1 (fr) 1992-05-25 1993-12-09 Centre National De La Recherche Scientifique (Cnrs) Composes biologiquement actifs de type phosphotriesters
WO1994026764A1 (fr) 1993-05-12 1994-11-24 Centre National De La Recherche Scientifique (Cnrs) Oligonucleotides phosphorothioates triesters et procede de preparation
WO1997030731A2 (fr) 1996-02-21 1997-08-28 The Immune Response Corporation Procede de preparation de complexes porteurs de polynucleotides pour administration a des cellules
US5705188A (en) 1993-02-19 1998-01-06 Nippon Shinyaku Company, Ltd. Drug composition containing nucleic acid copolymer
US20040102618A1 (en) 1999-09-30 2004-05-27 Isis Pharmaceuticals, Inc. Human RNase H1 and oligonucleotide compositions thereof
WO2004046160A2 (fr) 2002-11-18 2004-06-03 Santaris Pharma A/S Conception antisens
US20040235773A1 (en) 2003-04-13 2004-11-25 Hong Zhao Polymeric oligonucleotide prodrugs
US7087229B2 (en) 2003-05-30 2006-08-08 Enzon Pharmaceuticals, Inc. Releasable polymeric conjugates based on aliphatic biodegradable linkers
WO2007031091A2 (fr) 2005-09-15 2007-03-22 Santaris Pharma A/S Composes antagonistes d'arn de modulation de l'expression de p21 ras
WO2007134181A2 (fr) 2006-05-11 2007-11-22 Isis Pharmaceuticals, Inc. Analogues d'acides nucléiques bicycliques modifiés en 5'
WO2007146511A2 (fr) 2006-05-05 2007-12-21 Isis Pharmaceuticals, Inc. Composés et procédés de modulation de l'expression génique
WO2008034123A2 (fr) 2006-09-15 2008-03-20 Enzon Pharmaceuticals, Inc. Conjugués polymères contenant des fragments chargés positivement
US7399845B2 (en) 2006-01-27 2008-07-15 Isis Pharmaceuticals, Inc. 6-modified bicyclic nucleic acid analogs
WO2008113832A2 (fr) 2007-03-22 2008-09-25 Santaris Pharma A/S Composés arn antagonistes courts pour la modulation de l'arnm cible
WO2008150729A2 (fr) 2007-05-30 2008-12-11 Isis Pharmaceuticals, Inc. Analogues d'acides nucléiques bicycliques pontés par aminométhylène n-substitué
WO2008154401A2 (fr) 2007-06-08 2008-12-18 Isis Pharmaceuticals, Inc. Analogues d'acide nucléique bicyclique carbocylique
WO2009006478A2 (fr) 2007-07-05 2009-01-08 Isis Pharmaceuticals, Inc. Analogues d'acides nucléiques bicycliques disubstitués en position 6
WO2009067647A1 (fr) 2007-11-21 2009-05-28 Isis Pharmaceuticals, Inc. Analogues d'acide nucléique alpha-l-bicyclique carbocyclique
WO2009090182A1 (fr) 2008-01-14 2009-07-23 Santaris Pharma A/S Oligonucléotides de type « gapmère » de nucléotide d'adn substitué en c4'
WO2009100320A2 (fr) 2008-02-07 2009-08-13 Isis Pharmaceuticals, Inc. Analogues d’acides nucléiques de cyclohexitol bicycliques
WO2009124238A1 (fr) 2008-04-04 2009-10-08 Isis Pharmaceuticals, Inc. Composés oligomères comprenant des nucléosides bicycliques terminaux liés de façon neutre
WO2009124295A2 (fr) 2008-04-04 2009-10-08 Isis Pharmaceuticals, Inc. Composés oligomères comprenant des nucléosides bicycliques et ayant une toxicité réduite
WO2010036698A1 (fr) 2008-09-24 2010-04-01 Isis Pharmaceuticals, Inc. Nucléosides alpha-l-bicycliques substitués
WO2011017521A2 (fr) 2009-08-06 2011-02-10 Isis Pharmaceuticals, Inc. Analogues d'acides nucléiques cyclohexoses bicycliques
WO2011085102A1 (fr) 2010-01-11 2011-07-14 Isis Pharmaceuticals, Inc. Nucléosides bicycliques à base modifiée et composés oligomères préparés à partir de ceux-ci
WO2011115818A1 (fr) 2010-03-17 2011-09-22 Isis Pharmaceuticals, Inc. Nucléosides bicycliques 5'-substitués et composés oligomères synthétisés à partir desdits nucléosides
WO2014062686A1 (fr) * 2012-10-15 2014-04-24 Isis Pharmaceuticals, Inc. Procédés pour moduler l'expression de c90rf72
WO2014062736A1 (fr) * 2012-10-15 2014-04-24 Isis Pharmaceuticals, Inc. Procédés permettant de surveiller l'expression de c9orf72
WO2014062691A2 (fr) * 2012-10-15 2014-04-24 Isis Pharmaceuticals, Inc. Compositions permettant de moduler l'expression de c90rf72

Patent Citations (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4914210A (en) 1987-10-02 1990-04-03 Cetus Corporation Oligonucleotide functionalizing reagents
US4962029A (en) 1987-10-02 1990-10-09 Cetus Corporation Covalent oligonucleotide-horseradish peroxidase conjugate
WO1993024510A1 (fr) 1992-05-25 1993-12-09 Centre National De La Recherche Scientifique (Cnrs) Composes biologiquement actifs de type phosphotriesters
US5705188A (en) 1993-02-19 1998-01-06 Nippon Shinyaku Company, Ltd. Drug composition containing nucleic acid copolymer
WO1994026764A1 (fr) 1993-05-12 1994-11-24 Centre National De La Recherche Scientifique (Cnrs) Oligonucleotides phosphorothioates triesters et procede de preparation
US5770713A (en) 1993-05-12 1998-06-23 Centre National De La Recherche Scientifique Phosphorothioate triester oligonucleotides and method of preparation
WO1997030731A2 (fr) 1996-02-21 1997-08-28 The Immune Response Corporation Procede de preparation de complexes porteurs de polynucleotides pour administration a des cellules
US20040102618A1 (en) 1999-09-30 2004-05-27 Isis Pharmaceuticals, Inc. Human RNase H1 and oligonucleotide compositions thereof
WO2004046160A2 (fr) 2002-11-18 2004-06-03 Santaris Pharma A/S Conception antisens
US20040235773A1 (en) 2003-04-13 2004-11-25 Hong Zhao Polymeric oligonucleotide prodrugs
US7087229B2 (en) 2003-05-30 2006-08-08 Enzon Pharmaceuticals, Inc. Releasable polymeric conjugates based on aliphatic biodegradable linkers
WO2007031091A2 (fr) 2005-09-15 2007-03-22 Santaris Pharma A/S Composes antagonistes d'arn de modulation de l'expression de p21 ras
US7399845B2 (en) 2006-01-27 2008-07-15 Isis Pharmaceuticals, Inc. 6-modified bicyclic nucleic acid analogs
WO2007146511A2 (fr) 2006-05-05 2007-12-21 Isis Pharmaceuticals, Inc. Composés et procédés de modulation de l'expression génique
WO2007134181A2 (fr) 2006-05-11 2007-11-22 Isis Pharmaceuticals, Inc. Analogues d'acides nucléiques bicycliques modifiés en 5'
WO2008034123A2 (fr) 2006-09-15 2008-03-20 Enzon Pharmaceuticals, Inc. Conjugués polymères contenant des fragments chargés positivement
WO2008113832A2 (fr) 2007-03-22 2008-09-25 Santaris Pharma A/S Composés arn antagonistes courts pour la modulation de l'arnm cible
WO2008150729A2 (fr) 2007-05-30 2008-12-11 Isis Pharmaceuticals, Inc. Analogues d'acides nucléiques bicycliques pontés par aminométhylène n-substitué
WO2008154401A2 (fr) 2007-06-08 2008-12-18 Isis Pharmaceuticals, Inc. Analogues d'acide nucléique bicyclique carbocylique
WO2009006478A2 (fr) 2007-07-05 2009-01-08 Isis Pharmaceuticals, Inc. Analogues d'acides nucléiques bicycliques disubstitués en position 6
WO2009067647A1 (fr) 2007-11-21 2009-05-28 Isis Pharmaceuticals, Inc. Analogues d'acide nucléique alpha-l-bicyclique carbocyclique
WO2009090182A1 (fr) 2008-01-14 2009-07-23 Santaris Pharma A/S Oligonucléotides de type « gapmère » de nucléotide d'adn substitué en c4'
WO2009100320A2 (fr) 2008-02-07 2009-08-13 Isis Pharmaceuticals, Inc. Analogues d’acides nucléiques de cyclohexitol bicycliques
WO2009124238A1 (fr) 2008-04-04 2009-10-08 Isis Pharmaceuticals, Inc. Composés oligomères comprenant des nucléosides bicycliques terminaux liés de façon neutre
WO2009124295A2 (fr) 2008-04-04 2009-10-08 Isis Pharmaceuticals, Inc. Composés oligomères comprenant des nucléosides bicycliques et ayant une toxicité réduite
WO2010036698A1 (fr) 2008-09-24 2010-04-01 Isis Pharmaceuticals, Inc. Nucléosides alpha-l-bicycliques substitués
WO2011017521A2 (fr) 2009-08-06 2011-02-10 Isis Pharmaceuticals, Inc. Analogues d'acides nucléiques cyclohexoses bicycliques
WO2011085102A1 (fr) 2010-01-11 2011-07-14 Isis Pharmaceuticals, Inc. Nucléosides bicycliques à base modifiée et composés oligomères préparés à partir de ceux-ci
WO2011115818A1 (fr) 2010-03-17 2011-09-22 Isis Pharmaceuticals, Inc. Nucléosides bicycliques 5'-substitués et composés oligomères synthétisés à partir desdits nucléosides
WO2014062686A1 (fr) * 2012-10-15 2014-04-24 Isis Pharmaceuticals, Inc. Procédés pour moduler l'expression de c90rf72
WO2014062736A1 (fr) * 2012-10-15 2014-04-24 Isis Pharmaceuticals, Inc. Procédés permettant de surveiller l'expression de c9orf72
WO2014062691A2 (fr) * 2012-10-15 2014-04-24 Isis Pharmaceuticals, Inc. Compositions permettant de moduler l'expression de c90rf72

Non-Patent Citations (34)

* Cited by examiner, † Cited by third party
Title
ASH ET AL., NEURON, vol. 77, 2013, pages 639 - 646
BERGE ET AL.: "Pharmaceutical Salts", J. OF PHARM. SCI., vol. 66, 1977, pages 1 - 19
C. LAGIER-TOURENNE ET AL: "Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 110, no. 47, 19 November 2013 (2013-11-19), US, pages E4530 - E4539, XP055228419, ISSN: 0027-8424, DOI: 10.1073/pnas.1318835110 *
CARROLL, J. B. ET AL., MOL. THER., vol. 19, no. 12, 2011, pages 2178 - 2185
CHRISTENSEN, NUCL. ACIDS. RES., vol. 30, 2002, pages 4918 - 4925
D. SAREEN ET AL: "Targeting RNA Foci in iPSC-Derived Motor Neurons from ALS Patients with a C9ORF72 Repeat Expansion", SCIENCE TRANSLATIONAL MEDICINE, vol. 5, no. 208, 23 October 2013 (2013-10-23), Washington, DC, pages 208ra149 - 208ra149, XP055228414, ISSN: 1946-6234, DOI: 10.1126/scitranslmed.3007529 *
DEJESUS-HERNANDEZ ET AL., NEURON, vol. 72, 2011, pages 245 - 256
DONNELLY CHRISTOPHER J ET AL: "RNA Toxicity from the ALS/FTDC9ORF72Expansion Is Mitigated by Antisense Intervention", NEURON, CELL PRESS, US, vol. 80, no. 2, 7 October 2013 (2013-10-07), pages 415 - 428, XP028757398, ISSN: 0896-6273, DOI: 10.1016/J.NEURON.2013.10.015 *
DONNELLY ET AL., NEURON, vol. 80, 2013, pages 415 - 428
FARR ET AL., FREE RADIC BIOL MED., vol. 67, February 2014 (2014-02-01), pages 387 - 95
FARR ET AL., J ALZHEIMERS DIS., vol. 40, no. 4, 1 January 2014 (2014-01-01), pages 1005 - 16
FLUITER ET AL., MOL. BIOSYST., vol. 5, 2009, pages 838 - 843
FREIER; ALTMANN, NUCL. ACID RES., vol. 25, 1997, pages 4429 - 4443
FRIBERG ET AL., MOLECULAR THERAPY-NUCLEIC ACIDS, vol. E9, 2012, pages 1 - 12
GRAFF JR ET AL., J CLIN INVEST, vol. 117, 2007, pages 2638 - 2648
HERNANDEZ ET AL., NEURON, vol. 72, 2011, pages 245 - 256
LAGIER-TOURENNE, PNAS, vol. 110, no. 47, 2013, pages E4530 - E4539
LING ET AL., NEURON, vol. 79, 2011, pages 416 - 438
M. KOHJI ET AL., SCIENCE, vol. 339, no. 6125, 2013, pages 1335 - 1338
MANOHARAN ET AL., TETRAHEDRON LETTERS, vol. 32, 1991, pages 7171
MARTIN: "Remington's Pharmaceutical Sciences, 18th ed.", 1990, MACK PUBLISHING CO.
MORI ET AL., SCIENCE, vol. 339, 2013, pages 1335 - 1338
RENTON ET AL., NEURON, vol. 72, 2011, pages 257 - 268
SMITH ET AL., J. CLIN. INVEST., vol. 116, 2006, pages 2290 - 2296
SOIFER, H.S., METHODS MOL BIOL., vol. 815, 2012, pages 333 - 46
SORANA CIURA ET AL: "Loss of function of C9orf72 causes motor deficits in a zebrafish model of Amyotrophic Lateral Sclerosis", ANNALS OF NEUROLOGY., 1 April 2013 (2013-04-01), BOSTON, US, pages n/a - n/a, XP055228421, ISSN: 0364-5134, DOI: 10.1002/ana.23946 *
STEIN, C.A. ET AL., NUC. ACIDS RES., vol. 38, no. 1, 2010, pages 1 - 8
T. ZU ET AL., PNAS, vol. 110, no. 51, 2013, pages E4968 - E4977
THEODORA W GREENE; PETER G M WUTS: "Protective Groups in Organic Synthesis, 3rd ed.", 1999, JOHN WILEY & SONS
TODD PETER K ET AL: "C9orf72-Associated FTD/ALS: When Less Is More", NEURON, CELL PRESS, US, vol. 80, no. 2, 7 October 2013 (2013-10-07), pages 257 - 258, XP028757394, ISSN: 0896-6273, DOI: 10.1016/J.NEURON.2013.10.010 *
UHLMANN, CURR. OPINION IN DRUG DEVELOPMENT, vol. 3, no. 2, 2000, pages 293 - 213
VESTER ET AL., BIOORG. MED. CHEM. LETT., vol. 18, 2008, pages 2296 - 2300
ZHAO ET AL., BIOCONJUGATE CHEM., vol. 16, 2005, pages 758 - 766
ZHAO ET AL., J. CONTROLLED RELEASE, vol. 119, 2007, pages 143 - 152

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10443052B2 (en) 2012-10-15 2019-10-15 Ionis Pharmaceuticals, Inc. Compositions for modulating C9ORF72 expression
US10577604B2 (en) 2012-10-15 2020-03-03 Ionis Pharmaceuticals, Inc. Methods for monitoring C9ORF72 expression
US9963699B2 (en) 2012-10-15 2018-05-08 Ionis Pharmaceuticals, Inc. Methods for modulating C9ORF72 expression
US11339393B2 (en) 2013-10-11 2022-05-24 Ionis Pharmaceuticals, Inc. Compositions for modulating C9ORF72 expression
US10221414B2 (en) 2013-10-11 2019-03-05 Ionis Pharmaceuticals, Inc. Compositions for modulating C9ORF72 expression
US9605263B2 (en) 2015-04-16 2017-03-28 Ionis Pharmaceuticals, Inc. Compositions for modulating C9ORF72 expression
WO2017079291A1 (fr) * 2015-11-02 2017-05-11 Ionis Pharmaceuticals, Inc. Composés et méthodes de modulation de c90rf72
US11260073B2 (en) 2015-11-02 2022-03-01 Ionis Pharmaceuticals, Inc. Compounds and methods for modulating C90RF72
EP3664816A4 (fr) * 2017-08-08 2021-05-19 Wave Life Sciences Ltd. Compositions oligonucléotidiques et procédés associés
US11739325B2 (en) 2017-08-08 2023-08-29 Wave Life Sciences Ltd. Oligonucleotide compositions and methods thereof
EP4093409A4 (fr) * 2020-01-23 2024-01-10 University of Southern California Antagonisme en tant que thérapie pour des protéinopathies tdp-43
WO2021205005A3 (fr) * 2020-04-09 2021-11-18 Association Institut De Myologie Séquences antisens pour le traitement de la sclérose latérale amyotrophique
CN115516093A (zh) * 2020-04-09 2022-12-23 肌肉学研究协会 用于治疗肌萎缩侧索硬化症的反义序列
US20220290136A1 (en) * 2020-09-30 2022-09-15 Crispr Therapeutics Ag Materials and methods for treatment of amyotrophic lateral sclerosis

Also Published As

Publication number Publication date
US20160108396A1 (en) 2016-04-21
WO2016024205A8 (fr) 2016-10-20
CA2900208A1 (fr) 2016-02-15

Similar Documents

Publication Publication Date Title
US20160108396A1 (en) Oligomers targeting hexanucleotide repeat expansion in human c9orf72 gene
EP2152879B1 (fr) Composés antagonistes de l'arn pour la modulation de la béta caténine
US8268793B2 (en) RNA antagonist compounds for the modulation of HER3
CA3103756A1 (fr) Oligonucleotides pour moduler l'expression de scn9a
WO2016019063A1 (fr) Compositions et méthodes pour moduler l'expression de tau
US20110124709A1 (en) Rna antagonists targeting gli2
US20110172296A1 (en) Modulation of transforming growth factor-beta 1 expression
US20120115936A1 (en) Potent anti apob antisense compounds
US20100249219A1 (en) Short rna antagonist compounds for the modulation of hif-1alpha
JP2011505798A (ja) Ｍｃｌ−１を調節するためのＲＮＡアンタゴニスト化合物
US8440809B2 (en) RNA antagonists targeting Hsp27
US9040493B2 (en) RNA antagonists targeting GLI2 for the treatment of leukemia
US20140323557A1 (en) Compounds for the modulation of beta-catenin expression and uses thereof
US20110224281A1 (en) Rna antagonists targeting hsp70-2
EP2205738A2 (fr) Composés antagonistes de l'arn courts destinés à moduler hif1alpha
AU2014265070A1 (en) Rna antagonist compounds for the modulation of beta-catenin
WO2012066092A1 (fr) Composés de modulation de l'expression de l'aurora kinase a

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15774968

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15774968

Country of ref document: EP

Kind code of ref document: A1